Zanthoxylum schinifolium is sold at a higher price than other Zanthoxylum species and is frequently adulterated with closely related Zanthoxylum species because of its high demand as a Korean food ingredient and medicinal material in markets. In addition, the pericarps of three Zanthoxylum species  are defined as herbal medicine Zanthoxyli Pericarpium in Korean pharmacopoeias, but not Z. piperitum in Chinese pharmacopoeias. Further confusion arises in the morphological similarity between Z. armatum (adulterant) and Z. bungeanum. Therefore, the aim of this study was to develop a sequence characterized amplified region (SCAR) marker for discrimination of four Zanthoxylum species.To ensure the safety, quality and therapeutic efficacy of processed foods and herbal medicines, it is important to identify and discriminate economically motivated adulterants. Z. armatum are being distributed as Zanthoxyli Pericarpium in Korean and Chinese markets.With the goal of developing rapid and reliable tools for genetic discrimination of authentic Zanthoxyli Pericarpium, we designed species‐specific SCAR markers, based on ITS2 sequences, that generate amplicons of less than 200 bp. Using these markers, we established both conventional and real‐time PCR assay methods capable of differentiating samples at the species level. We validated the ability of SCAR markers to authenticate edible oil and herbal medicine, and confirmed that some herbal medicines contaminated with Journal of the Science of Food and Agriculture published by John Wiley & Sons Ltd on behalf of Society of Chemical Industry.The SCAR markers and PCR methods described represent powerful tools for protecting against adulteration and ensuring standardization of processed foods and herbal medicine. © 2018 The Authors.  Adulteration or contamination of food and herbal medicines with inauthentic materials affects safety, price and taste, as well as pharmaceutical properties and nutrient quality. Accordingly, certification of authenticity is very important in both contexts.matK and rbcL, recommended by the Plant Working Group of the Consortium for the Barcode of Life (CBOL), DNA barcoding of regions such as ITS , psbA‐trnH, trnL‐F and rps16 has been used to identify species in plant taxa.Molecular markers at the DNA level offer cheap, rapid and accurate analysis.Raw food materials are altered by various manufacturing techniques involving physical, chemical or biochemical treatments.Zanthoxylum (Rutaceae) consists of approximately 250 species worldwide, of which six are distributed in Korea.Zanthoxylum species, Z. schinifolium, Z. piperitum and Z. armatum, are distributed throughout the Korean Peninsula and traditionally have been used for food and medicinal materials. However, Z. coreanum, Z. fauriei and Z. ailanthoides have not been used for this purpose because these species are distributed at restricted regions such as Jeju Island or the southern seaside part of the Korean Peninsula. Especially, Z. schinifolium (Chinese pepper) has been used as a spice for a long time in Korea, as well as in other Asian countries.Zanthoxylum species, and is also used in folk remedies to treat stomachache and asthma. On the other hand, Z. bungeanum (Sichuan pepper) is not distributed in Korea, but is an indispensable spice and herbal medicine in China.The genus Zanthoxylum species are used as herbal medicines as well as food materials. Zanthoxyli Pericarpium, the name of a herbal medicine, is prescribed to treat abdominal pain, toothache, dyspepsia, vomiting, diarrhea, ascariasis and eczema in traditional Korean and Chinese medicine, but these national pharmacopoeias differ in how they define botanical species.Z. schinifolium, Z. piperitum or Z. bungeanum are defined as the authentic herbal medicine Zanthoxyli Pericarpium, whereas the Chinese pharmacopoeia describes only two species, Z. schinifolium or Z. bungeanum.Z. armatum, an inauthentic adulterant species, are very similar to those of authentic Z. bungeanum. Therefore, the identification of authentic material is necessary for quality control and prevention of adulteration.Some Zanthoxylum species. Candidate SCAR markers, designed using species‐specific sequences from 22 samples, were validated for the establishment of conventional and real‐time PCR assays using SYBR Green. We validated the species discrimination ability of these approaches using the SCAR markers we developed to authenticate edible oil and commercial herbal medicines.In this study, we analyzed the DNA barcode sequences using ITS2 universal primers for four Zanthoxylum species utilized as food ingredients or herbal medicines, Z. schinifolium, Z. piperitum, Z. armatum and Z. bungeanum, were subjected to analysis of DNA barcode sequences and used to establish PCR assay methods . Prior to extraction using the kit, approximately 100 mg of sample in 1.6 mL of AP1 buffer was completely crushed using a Precellys™ grinder , as described previously.A260/A280 ratio of 1.8–2.0 was used as a template for standard curve analysis in real‐time PCR. All samples were diluted to a final concentration of ca 15 ng µL−1.For the SCAR marker and real‐time PCR assays, genomic DNA of 22 samples was prepared using the DNeasyTo concentrate Chinese pepper oil, 50 mL of sample was centrifuged at 3000 rpm for 20 min. After centrifugation, the supernatant was discarded and 1 mL of Lysis Buffer L of the Olive Oil DNA Isolation Kit was added to the pellet . The pellet was resuspended vigorously using a vortex for 60 s, and incubated at 65 °C for 10 min. To extract genomic DNA, subsequent steps were performed according to the manufacturer's instructions.–1 ITS2‐S2F and ITS4 primers, and Solg™ 2× Taq PCR Smart‐Mix I . PCR conditions were as follows: initial denaturation at 95 °C for 2 min; followed by 35 cycles of denaturation at 95 °C for 40 s, annealing at 53 °C for 40 s and elongation at 72 °C for 1 min; and a final elongation at 72 °C for 5 min. The amplified DNA fragments were subjected to electrophoresis, and then extracted from the agarose gel using the Gel Extraction Kit (Qiagen). The eluted DNA fragments were ligated into the pGEM®‐T Easy Vector , and transformed into Escherichia coli competent cells using HIT™‐DH5‐α Value 108 . The recombinant E. coli were incubated for 18 h at 37 °C, and then selected in the presence of 100 µg mL–1 ampicillin, 40 µg mL–1 X‐gal and 0.5 mmol L–1 IPTG on LB agar medium. To confirm the presence of the representative sequence, six white colonies were analyzed per sample. The insert regions of plasmid DNA were sequenced using dideoxynucleotide chain termination sequencing (Sanger sequencing) with standard SP6 and T7 primers. Raw ITS2 sequences were edited using the BioEdit software (version 7.2.5) to trim unnecessary sequences, and then sequences of individual samples were aligned by ClustalW multiple alignment.ITS2 was amplified from 22 samples using universal primers ITS2‐S2F (5′‐ATG CGA TAC TTG GTG TGA AT‐3′) and ITS4 (5′‐TCC TCC GCT TAT TGA TAT GC‐3′).Zanthoxylum species. Several candidate primers were selected based on species‐specific substitutions; the number of combinations was 4 in Z. schinifolium, 20 in Z. piperitum, 4 in Z. armatum and 28 in Z. bungeanum. To verify primer specificity, conventional PCR was performed in a total reaction volume of 25 µL, containing approximately 15 ng of template, 0.4 µmol L–1 of each forward and reverse primer and Solg™ 2× Taq PCR Smart‐Mix I (Solgent). SCAR marker assays were conducted with a Proflex PCR system  with the following conditions: initial denaturation at 95 °C for 2 min; 35 cycles of denaturation at 95 °C for 20 s, annealing at 63 °C for 30 s and elongation at 72 °C for 20 s; and final elongation at 72 °C for 5 min. After PCR, products and 100 bp DNA ladder (Solgent) were separated on a 1.5% agarose gel in 0.5× Tris acetate–EDTA buffer as a running buffer.Species‐specific primers used for the SCAR marker assay were designed based on the variability of ITS2 sequences in four –1 each of forward and reverse primers and QuantiNova™ SYBR Green PCR Master Mix (Qiagen). To prepare a standard curve, species‐specific DNA fragments subcloned into the pGEM®‐T Easy Vector (Promega) were used as template. Species‐specific DNA fragments of a serially diluted template  were used for quantitation and to confirm the efficiency of equivalent amplification.R2 coefficient, slope (S) and amplification efficiency were calculated using the standard curve calibrated from the serially diluted template. The limit of quantitation (LOQ) and LOD were respectively obtained from 15 and 60 replicates using species‐specific DNA fragments as templates , according to the guidelines of Minimum Information for Publication of Quantitative Real‐Time PCR Experiments and the Joint Research Centre (JRC).Real‐time PCR was performed in a total reaction volume of 25 µL, containing approximately 15 ng of template, 0.4 µmol LZ. schinifolium farm were purchased. All edible Chinese pepper oil used in this study was extracted by the expeller or oil‐press methods. Validation and monitoring using the conventional and real‐time PCR assays were carried out as described above using the four species‐specific SCAR markers.To test the species discrimination abilities of the conventional and real‐time PCR assays, 10 herbal medicines distributed as Zanthoxyli Pericarpium from herbal markets in Korea and China and 12 samples of edible Chinese pepper oil produced at a Korean Efficiency of PCR amplification is an important factor in the evaluation of DNA barcoding regions.Zanthoxylum species could be successfully amplified with the ITS2‐SF and ITS4 universal primers. The trustworthy sequences of individual samples obtained with the T‐vector cloning system have been submitted to the GenBank database; accession numbers are listed in Table Z. schinifolium, Z. piperitum, Z. armatum and Z. bungeanum were 385, 383, 388 and 385 bases, respectively. Multiple alignment of these 22 sequences of the ITS2 region from four Zanthoxylum species revealed high identity , but ITS2 exhibited more species‐specific sequence variation than those two loci, and was therefore most suitable for developing SCAR markers for rapid and accurate discrimination of the four Zanthoxylum species.We confirmed that the 22 samples of four Zanthoxylum species . Detailed information about the primers used in this study is provided in Table Zanthoxylum species are shown in Fig. Z. schinifolium, but not from the other three species. In addition, species‐specific amplicons were obtained from Z. piperitum (139 bp), Z. armatum (111 bp) and Z. bungeanum (100 bp).To develop the species‐specific SCAR marker, we screened candidate primers: 4 combinations for Zanthoxylum species in herbal medicines or processed foods, including fermentation food and additives. Analysis of the PCR products obtained using the species‐specific SCAR markers revealed that their sequences were identical in each of the Zanthoxylum species tested. To test cross‐reactivity with other species or organisms, 26 samples comprising 6 vegetable oil‐related species , 4 processed food‐related species  and 1 herbal medicine‐related species (Ophiocordyceps sinensis) were amplified using four species‐specific SCAR markers , and exists commonly in microorganisms, animals and fungi as well as in plant taxa.–1) , efficiency (E) and R2 were calculated from the standard curve  between Ct values and template concentration, which should be greater than 0.98 in a reliable method, was 0.99629 in Z. schinifolium, 0.99908 in Z. piperitum, 0.99485 in Z. armatum and 0.99652 in Z. bungeanum. Thus, for all four Zanthoxylum species, real‐time PCR was highly sensitive and linear in concentration ranges from 108 to 103 copies  and Z. bungeanum (authentic).Zanthoxylum species. In addition, these primers produced amplicons of small size that are easily detected even when DNA has been highly fragmented during processing.Due to high demand for Korean food and medicine, Z. schinifolium farm, and then tested genetic discrimination using the conventional PCR and real‐time PCR assays.Next, we tested the ability of the species‐specific SCAR markers to genetically discriminate DNA extracted from edible oil. Edible oil extraction methods include expeller and oil press. Given the pharmacological effect of Zanthoxyli Pericarpium, some Chinese pepper oil is manufactured from seed containing the pericarp.Z. schinifolium  were identified as Z. bungeanum. Although it is not included in Chinese pharmacopoeias, Z. piperitum was identified in one sample (no. 15). Also, one sample of Zanthoxyli Pericarpium (no. 14) from a Chinese herbal market was identified as Z. schinifolium mixed with Z. armatum. Thus, we confirmed that adulterants of Zanthoxyli Pericarpium were distributed in Korean herbal markets. To compare the analytic reproducibility of the real‐time PCR assay using the SCAR marker with the conventional PCR assay, we carried out species discrimination assays using the 12 edible oil samples and 10 commercial herbal medicines. The real‐time PCR results for all samples were consistent with the conventional PCR results , Z. piperitum, Z. armatum and Z. bungeanum (Sichuan pepper), which are used in processed food and herbal medicines. We then designed SCAR markers based on the species‐specific sequence substitutions in the ITS2 locus. The SCAR markers were designed to generate amplicons shorter than 200 bp, which are advantageous for genetic discrimination of damaged DNA present in processed food and herbal medicines. The SCAR markers were suitable for both conventional and real‐time PCR assays, and had high detection sensitivity (at the picogram level). In addition, we validated the SCAR markers by analyzing commercial products, including 12 edible Chinese pepper oils and 10 commercial herbal medicines. The results revealed that some commercial herbal medicines are mixtures of two or more pericarps of Zanthoxylum species, and that Zanthoxyli Pericarpium is contaminated with inauthentic adulterants such as Z. armatum. Thus, the SCAR marker‐based PCR assays developed in this study provide useful genetic tools for quality control and standardization of food and herbal medicines.We analyzed the ITS2 sequence of four Figure S1. Multiple alignment of ITS2 sequences and positions of SCAR primers. Boxes indicate primer sequences and orientations.Click here for additional data file.Figure S2. Verification of the specificity of the SCAR markers in conventional PCR assays, using 26 samples of oil related plant species and other organisms. Lanes C1–8: Control plant samples: Z. schinifolium ZS_SC and SGP, Z. piperitum ZP_BU and GY, Z. armatum ZA_UJ and GS, and Z. bungeanum ZB_JK and SC#2. Lanes 1–27: plant species used as vegetable oil: G. max (soybean), B. napus (rape), Z. mays (corn), V. vinifera (Grape), S. indicum (sesame), P. frutescens (perilla). Lanes 18–26: fungus species used as food supplements or herbal medicines: C. militaris (Militaris Dong Chung Ha Cho), I. tenuipes (Dong Chung Ha Cho), O. sinensis (Cordyceps), S. sanghuang (Sang Hwang Beo Seot), S. baumii (Jang Su Jin Heut Beo Seot). (A–D) PCR amplification of SCAR markers specific for (A) Z. schinifolium (B) Z. piperitum, (C) Z. armatum, and (D) Z. bungeanum.Click here for additional data file.Figure S3. Verification of the specificity of SCAR markers in real‐time PCR, using 22 samples of four Zanthoxylum species. (A–D) PCR cycling SCAR markers specific for (A) Z. schinifolium, (B) Z. piperitum, (C) Z. armatum, and (D) Z. bungeanum. Arrows indicate PCR amplification of the species‐specific SCAR marker in the target species and the other three Zanthoxylum species. Threshold was determined manually using Rotor‐Gene Q software (version 2.3.1), based on standard curve analysis.Click here for additional data file.Table S1. List of oil related plant species and other organisms analyzed in this study to validate the specificity of the SCAR markers.Table S2. Optimization according to primer concentration, and temperature or time for annealing‐extension.Table S3. Estimation of LOQ and LOD of the real‐time PCR assay.Click here for additional data file.


Interaural phase difference (IPD) discrimination upper frequency limits andjust-noticeable differences (JNDs), interaural level difference (ILD) JNDs, anddiotic intensity JNDs were measured for 20 older hearing-impaired listeners withmatched moderate sloping to severe sensorineural hearing losses. The JNDs weremeasured using tone stimuli at 500 Hz. In addition to these auditory tests, theparticipants completed a cognitive test (Trail Making Test). Significantperformance improvements in IPD discrimination were observed across testsessions. Strong correlations were found between IPD and ILD discriminationperformance. Very strong correlations were observed between IPD discriminationand Trail Making performance as well as strong correlations between ILDdiscrimination and Trail Making performance. These relationships indicate thatinterindividual variability in IPD discrimination performance did notexclusively reflect deficits specific to any auditory processing, includingearly auditory processing of temporal information. The observed relationshipsbetween spatial audition and cognition may instead be attributable to amodality-general spatial processing deficit and/or individual differences inglobal processing speed. The discrimination of interaural time differences (ITDs) and interaural phasedifferences (IPDs) are suprathreshold functions of binaural hearing that facilitatethe localization of sound sources in the horizontal plane. Hearing-impaired (HI)listeners can show poorer than normal ITD and IPD discrimination performance even inthe absence of a pure-tone hearing loss at the test frequencies e.g., . For thiInterindividual variability in IPD discrimination performance among HI listeners hasbeen found to be, at best, moderately associated with pure-tone hearing thresholds. FurtherAt the level of the basilar membrane, sound signals can be analyzed in terms of anenvelope imposed on a rapidly oscillating carrier, referred to as temporal finestructure TFS; . InformaThe idea for this study arose from observations we made in a previous, unpublishedstudy. Among other psychoacoustic tests in that study, we measured IPDdiscrimination upper frequency limits (FLs) using tone sequence stimuli similar tothose used by We measured IPD discrimination FLs and just-noticeable differences (JNDs) repeatedlyacross test sessions to explore potential training effects. Furthermore, we includedintensity discrimination and ILD discrimination tasks to examine whether listenerswho show difficulties with IPD discrimination would also experience difficultieswith these tasks as suggested by previous studies . In addiLF), ranged from 37 to52 dB HL. All ears showed clear ear canals under otoscopic inspection, andair-bone gaps were smaller than or equal to 10 dB, except for participants f9and m2. Participant f9 showed an air-bone gap of 15 dB at 500 Hz in the rightear, and m2 showed air-bone gaps of 20 dB at 125 and 250 Hz in both ears.Furthermore, m2 was the only participant who reported having had ear tubesinserted at age 8 years. Thus, participants f9 and m2 may have had mixed hearinglosses with small conductive components in the low frequencies, while theremaining participants showed hearing losses of purely sensorineural origin.The 20 HI participants (11 women and nine men) were aged between 48 and 85 years(median: 71 years). Their demographic and audiologic information is detailed inParticipants were paid $20 per hour. The study was approved by the BiomedicalInstitutional Review Board of the University of Louisville.A version of the MoCA for the hearing impaired with visual instructions wasadministered nonverbally using a timed PowerPoint (Microsoft Corp.)presentation . The MoCPart A of the Trail Making Test   was admiParticipants first performed a TMA practice run on a shorter layoutconsisting of eight circles before performing the 25-circle TMA test.Subsequently, they performed a short practice run of TMB followed by thefull 25-circle TMB test. In contrast to the MoCA Trail Making subtask, theTrail Making Test was timed, and the test score was the time to complete thetrail starting from when the circles were first displayed on the screen.Participants could only fail the TMA or TMB tests if they were not able tocomplete each trail within the allowed maximum time of 300 seconds, at whichpoint the tests were automatically aborted cf. .In addition to the TMA and TMB times, we calculated the difference scoreTMB–TMA, which has been considered to be an indicator of executive controlabilities .We used a two-interval, two-alternative, forced-choice (2I-2AFC) task similarto the TFS test described by Participants were instructed to identify the interval in which the soundswere perceived to move (see Appendix A for detailed participantinstructions). The intervals were visually marked during playback byflashing boxes on a touchscreen display (GeChic 1002 10.1-in.) placed infront of the participants, and they responded by touching the correspondingbox. Tone frequency was varied adaptively from trial to trial using theBayes Fisher information gain method described by Similar to the procedures in psignifit 4 MATLAB toolbox for Bayesian PF estimation with negative slope and fitted using thetimation. The IPDThe 2I-2AFC measurement of IPD JND was very similar to the measurement of IPDdiscrimination FL. The main difference in the IPD JND measurement was thatthe tone frequency was fixed at 500 Hz, while the IPDs of the second andfourth tones in the target interval were varied adaptively to measurediscrimination performance as a function of IPD. The IPDs of the second andfourth tones had opposite signs, such that the second tone was lateralizedtoward the left and the fourth tone was lateralized toward the right side.This was done to provide participants with movement cues to both sides,since the side to which the target tones with 180° IPDs in the IPD FLmeasurement were lateralized was ambiguous. The set of possible IPDs in theadaptive run  were 50 Participants performed a training run consisting of 10 trials beforeperforming a measurement run of 60 trials. In contrast to the measurementsof IPD discrimination FL, ILD training runs were not included in the IPD JNDmeasurements. IPD discrimination performance as a function oflog-transformed IPD was fitted using a logistic PF model , and theSimilar to the IPD JND measurement, the ILD JND measurement was alsoperformed at a fixed frequency of 500 Hz. The main difference was that ILDswere used to induce movement instead of IPDs. In the reference interval, alltones had a 0-dB ILD. In the target interval, the first and third tones alsohad 0-dB ILDs, while the second and fourth tones had a finite ILD that wasvaried adaptively. The ILD was applied by lowering the tone level on theleft by ILD/2 and by increasing it on the right by ILD/2. Thus, both thesecond and fourth tones were lateralized toward the right. The set ofpossible ILDs in the adaptive run  were 80ILD JNDs were measured in three different experimental conditions similar tothose used by L. R. Tone levels (without ILDs and level roving) varied from 28 to 30 dB SL acrosstrials for all participants but m2 and f7. For m2 and f7, tone levels onsome trials were lowered to 25 and 22 dB SL, respectively, to limit themaximum presentation level to 95 dBA.Order effects might have complicated comparisons of individual performance ifthe three ILD discrimination conditions had been run block-wise. Therefore,the conditions were interleaved on a trial-by-trial basis, that is, thethree adaptive runs were performed at the same time. Since the imposed ILDsshifted both the second and fourth tones to the right side, participantswere instructed to identify the interval in which both tones moved to theright (see Appendix A). Participants performed a short No Rove training runconsisting of 6 trials followed by interleaved training runs of all threeconditions of 10 trials each, before performing the three interleavedmeasurement runs of 50 trials each.ILD discrimination performance as a function of log-transformed ILD wasfitted using a Weibull PF model , and theThe 2I-2AFC measurement of intensity JND (INT JND) at 500 Hz used sequencesof diotic tones without any binaural cues. All tones in the referenceinterval had identical levels, and the same level was applied to the firstand third tones of the target interval. The second and fourth tones of thetarget interval were presented at a higher level. The level incrementrelative to the other tones was varied adaptively. The set of possible levelincrements were 47 logarithmically scaled values from 0.1 to 10 dB, and allruns started with an increment of 6 dB. Tone levels (before application ofthe level increment) were 30 dB SL for all participants.Participants were instructed to detect the interval in which the tones variedin loudness (see Appendix A). They performed a training run of 10 trialsbefore performing the measurement run of 60 trials. Discriminationperformance as a function of log-transformed level increment was fittedusing a Weibull PF model  and the The psychoacoustic tests were performed in a sound-treated room at the HeuserHearing Institute. All stimuli were generated in MATLAB at a sampling rateof 48 kHz, converted to analog signals using an RME Fireface UCX audiointerface with 24-bit digital-to-analog conversion, and presented viaSennheiser HD 600 headphones. Stimulus presentation levels were calculatedby adding sensation levels to interpolated audiometric thresholds  andconverting dB HL to dB sound pressure level in an artificial ear (GRAS 43AA)using the probe-tube method described in Testing was conducted in two sessions of 1- to 2-hour duration each. Afterinformed consent and audiological examination, the MoCA was administered.Testing then proceeded in the first session with four psychoacoustic testsin the following order: IPD FL, ILD JND, IPD JND, and IPD FL. Testingresumed in the second session with the following tests: IPD FL, INT JND, IPDJND, and IPD FL. Thus, to explore potential practice effects, the IPD FL wasmeasured four times (each time preceded by ILD and IPD training as describedabove) and the IPD JND was measured twice. The second session concluded withthe Trail Making Test. The time intervals between the first and secondsession varied across participants from a single day to 7 days (median of 3days), with the exception of participant f9, who performed the secondsession 84 days after the first.F tests withKenward–Roger approximation for denominator degrees of freedom  were performed on general linearmixed-effects models GLMMs;  with mod freedom . Least-s freedom .The proedom . Three of the 20 participants  failed to successfully draw the alternating trail without (uncorrected)errors in the untimed TMB subtask of the MoCA.The MoCA scores for all participants are listed in SD = 20)seconds and 75 (SD = 40) seconds, respectively. IndividualTMA and TMB times ranged from 17 to 88 seconds and 31 to 160 seconds,respectively. The difference score TMB–TMA had an average value of 37(SD = 25) and ranged from 4 to 89 seconds.All participants completed the timed TMA and TMB tests in less than 300seconds. The average TMA and TMB times were 38  = 2.98, p = .04,with average performance continuously improving .Post hoc tests revealed significant differences between measurements 1 and 4as well as between measurements 2 and 4. F = 6.82,p = .02, with a lower average IPD JND in the secondmeasurement (22°) than in the first measurement (35°). F = 5.12,p = .01, with significantly lower ILD JNDs(p < .05) in the No Rove condition (2.8 dB) than inthe Level Rove condition (3.9 dB) and IPD/Level Rove condition (4.0 dB).However, measurement condition was no longer significant when the ILD JNDsof participants f9, m12, m16, and f17 were excluded from the model,F = 1.43, p = .25. In thiscase, the mean ILD JNDs were 2.5, 3.0, and 3.0 dB for the No Rove, LevelRove, and IPD/Level Rove conditions, respectively. Individual ILD JNDs for the three measurement conditions No Rove, Level Rove,and IPD/Level Rove are shown in SD = 0.6) dB. All participantsshowed similar performance without any outliers, as reflected by the smallstandard deviation. Individual INT JNDs ranged from 0.2 to 2.5 dB.The average INT JND was 1.2 . Thus, the ILD JND in the LevelRove condition was the best measure of spatial discriminationbased on binaural ILD cues. Log-transformed, normally distributed TMA and TMBtimes, IPD JNDs, ILD JNDs, and INT JNDs were used in the computation of thecorrelation coefficients. The resulting correlation matrix is listed in LF were partialled out  = 2.96,p = .04, TMB time was a significant predictor,F = 48.5, p < .0001. Theinteraction of TMB time with measurement number was not significant(p = .28). p > .3). They were, however, both significant(p < .01) when TMB time was deliberately excluded.Effects of age, PTALF, MoCA score, tinnitus, and musical trainingwere not significant (p > .17). Together, the fixedeffects measurement number and TMB time explained 63% of the variance. To explore significant predictors of IPD FL, we performed ANCOVAs with IPD FLas the dependent variable. The tested covariates, in addition to measurementnumber, were age, PTATo explore the extent to which TMB time mediated effects of age on IPD FL, weexamined a path model with age as an independent variable, IPD FL as thedependent variable, and TMB time as the mediator . The modF = 5.67,p = .03, TMB time was a significant predictor,F = 31.9, p < .0001. IPDJNDs increased with increasing TMB time. The interaction with measurementnumber was not significant (p = .15). Similar to the IPD FLresults, TMA time and the difference score TMB–TMA were not significant inthe model with TMB time (p > .4), but were bothsignificant (p < .05) when TMB time was deliberatelyexcluded from the model. Effects of age, PTALF, MoCA score,tinnitus, and musical training were not significant(p > .3). The fixed effects measurement number and TMBtime explained 54% of the variance.We performed ANCOVAs with IPD JND as the dependent variable and tested thesame covariates as in the preceding subsection. In addition to thesignificant effect of measurement number, F = 5.12,p = .01, MoCA score, F = 7.28,p = .02, and TMA time, F = 13.8, p = .002. ILD JND increased with increasing TMAtime and decreased with increasing MoCA score. Interaction terms were notsignificant (p > .5). Furthermore, TMB time was notsignificant (p = .81) when TMA time was included in themodel but was significant (p = .004) when TMA time wasdeliberately excluded. The difference score TMB–TMA was not significant,regardless of whether TMA time was included or not(p > .3). Furthermore, effects of age, PTALF,tinnitus, and musical training were not significant(p > .14). The fixed effects measurement condition, TMAtime, and MoCA score together explained 54% of the variance.ANCOVAs with ILD JND as the dependent variable revealed significant effectsof measurement condition, Overall, the results of the individual cognitive and psychoacoustical tests reportedin this work are consistent with the literature. Details of this consistency arediscussed in the subsections that follow. The practice effects observed for somelisteners in the IPD discrimination tasks are also discussed. Most important,however, were the clear and consistent relationships observed between primarymeasures, including IPD discrimination, ILD discrimination, and Trail Making. Theserelationships suggest that in an older HI population, IPD discrimination performancedoes not reflect TFS sensitivity per se, but must be influenced by factors that arenot specifically auditory and may be of higher order. These possibilities arediscussed with reference to a conceptual model.The MoCA scores of our study participants were very similar in terms of mean andstandard deviation to those reported in previous studies with HI participants.The Trail Making Test scores obtained in our study cannot easily be compared inabsolute terms with the literature since we used a touchscreen version of theTrail Making Test, with identical but mirrored trails for part A and B.Nevertheless, the observed mean TMA and TMB times were similar to those reportedby SD = 257) Hz observed inthis study was consistent with the mean IPD FLs ranging from 583 to 808 Hz inprevious studies that used similar tone sequence stimuli  was observed betweenIPD FL and age, which was no longer significant when the multiple testingcorrection was applied. Furthermore, the correlation between IPD JND and age(r = .37) was not significant, and no significant effect ofage was observed in any of the statistical models. Although low powercontributed to the lack of significance (the sample size of 20 participantsresulted in a power of 0.8 for detecting associations ofr = .58 or stronger), the strength of the observed agecorrelations was nevertheless consistent with previous studies that measured IPDFLs and IPD JNDs in older listeners with and without low-frequency hearinglosses  as wellas low power. Nevertheless, the observed weak strength of correlations wasconsistent with previous reports of weak correlations between IPD FL, IPD JND,ILD JND, and pure-tone thresholds or PTALF (The correlations with PTAor PTALF . Howeveror PTALF .r = −.89),reflecting the similarity of these measures in assessing IPD discrimination, and TMB time was also the strongest predictor in the respectivestatistical models. Besides TMB time, significant correlations were alsoobserved with TMA time and the difference score TMB–TMA . Interestingly, ILD JND was alsosignificantly correlated with TMA time (r = .74), IPD FL(r = −.70), and IPD JND (r = .66). Inaddition to TMA time, statistical modeling showed that the MoCA score was also asignificant predictor of ILD JND.IPD FL was very strongly correlated with IPD JND . When you tap the correct circle, a connecting line will bedrawn. If you do not tap the correct circle, no line will be drawn. The testis timed. Work as fast as you can. When you are ready, tap the screen tobegin a practice run.On the next screen, you will see circles with numbers and letters. Begin bytapping number 1, followed by A, then 2, then B, then 3, then C, and so on,until you reach the end (1-A-2-B-3-C…). When you tap the correct circle, aconnecting line will be drawn. If you do not tap the correct circle, no linewill be drawn. The test is timed. Work as fast as you can. When you areready, tap the screen to begin a practice run.In this measurement, we test how well you can hear certain differences betweensounds. You will be listening to tones presented over headphones. On each trial,you will hear two tone sequences:beep beep beep beep  beep beep beep beepWhile they are playing, the first tone sequence and second tone sequence arevisually highlighted on the touchscreen in front of you by the labels “1st” and“2nd,” respectively. Please take a look at the screen now.positions. This is what you need to listen for: One tonesequence will be positioned unmoving in the center of your head. It sounds likemono presentation. In the other tone sequence, movement willoccur: The tones will move repeatedly from the center to the side of your head.This may sound like going from mono to stereo. For example:The first and second tone sequences differ in terms of their             beep   beepbeep beep beep beep  beep   beepYour task is to identify the tone sequence where the movementoccurred. Whether the movement will occur in the first or secondtone sequence will vary randomly from trial to trial. If you heard movement inthe first sequence …  beep   beepbeep   beep      beep beep beep beep      Press “1st”If you heard movement in the second sequence …              beep    beepbeep beep beep beep   beep    beep        Press “2nd”The task will vary in difficulty from trial to trial. If you are not able to tellwhether movement occurred in the first or second tone sequence, just guess oneof the two (“1st” or “2nd”).After you enter your response, you will receive feedback: The correct responsewill be highlighted in green. Please pay attention to this feedback. We willbegin with a training run.The next measurement is very similar to the preceding measurements. Again, youneed to listen for movement in the tone sequences.Your task is to detect the tone sequence where both tones move to theright side.listen for movement and indicate whetherit was the first or second tone sequence in which both tones moved to the rightside.Note, the tones may vary in loudness. However, you cannot accomplish the task byfocusing on the loudness of the tones because these are random loudnessvariations. Instead, After you enter your response, you will receive feedback: The correct responsewill be highlighted in green. Please pay attention to this feedback. We willbegin with a training run.In the preceding measurements, you listened for movement. In contrast, in thenext measurement you need to listen for changes in loudness.Your task is to detect the tone sequence in which the tones varied inloudness. For example:beep beep beep beep   beep BEEP beep BEEP      Press “2nd”The task will vary in difficulty from trial to trial.After you enter your response, you will receive feedback: The correct responsewill be highlighted in green. Please pay attention to this feedback. We willbegin with a training run.


Tight control management of Crohn’s disease (CD) based on biomarkers is more effective than conventional clinical management; however, fecal calprotectin is not allowed in Asian and some Western countries. To investigate whether tight control management based on readily available serum biomarkers results in better outcomes, we retrospectively reviewed treatment courses of consecutive Japanese CD patients treated with anti-tumor necrosis factor agents between 2003 and 2018. The association between failure of tight control (C-reactive protein (CRP) ≥ 0.5 mg/dL or albumin (Alb) < 3.8 g/dL at week 8 or 24) and subsequent major adverse outcomes  were analyzed. Among 223 patients followed for >8 weeks, 88 patients experienced MAOs. Multivariate analysis identified penetrating type, CRP ≥ 0.5 mg/dL and Alb < 3.8 g/dL at week 8 as independent risk factors . Among 204 patients followed for >24 weeks, 80 patients experienced MAOs. Penetrating type, CRP ≥ 0.5 mg/dL, and Alb < 3.8 g/dL at week 24 were identified as independent risk factors . Even in settings without fecal calprotectin, tight control management based on serum CRP and Alb may help avoid MAOs. The conventional treatment approach guided by clinical symptoms may lead to unfavorable prognosis and adverse outcomes such as stenosis or fistula. These intestinal complications necessitate surgery in a large proportion of patients; cumulative surgery rates of 16.3% at 1 year, 33.3% at 5 years, and 46.6% at 10 years following diagnosis have been reported3. Mucosal healing was shown to be associated with lower cumulative surgery rate and better prognosis4. Disease monitoring and treatment optimization aimed at mucosal healing may improve disease prognosis. Based on these reports, a treat-to-target approach for CD has been advocated as a strategy to improve prognosis and minimize the need for surgery; this approach entails the use of both clinical and endoscopic remission as indicators5. However, in contrast to patients with ulcerative colitis in whom lesions are limited to colorectum and sigmoidoscopy is enough for assessing disease activity, it is not easy to perform frequent ileocolonoscopy or enteroscopy in patients with CD who have intermittent and sometimes extensive lesions throughout the intestines. Therefore, biomarker remission, as indicated by normalization of C-reactive protein (CRP) and fecal calprotectin, was considered as an adjunctive or alternative target5.Recent studies have identified mucosal healing as an important marker of disease activity and prognosis6. In the clinical management group, the treatment strategy was based on clinical symptoms only, while patients in the tight control group were managed based on clinical symptoms together with serum or fecal biomarkers; treatment failure criteria was set at CRP > 5 mg/L or fecal calprotectin >250 µg/g. After 48 weeks, tight control management was found to be more effective than conventional clinical management. In that study, fecal calprotectin was the most important index and in >90% of cases, treatment intensification was based on fecal calprotectin exceeding the treatment failure criteria. However, there is a need to develop additional biomarkers because fecal calprotectin is not always readily available in routine clinical practice in Asian and some Western countries. Serum CRP has been shown to be associated with disease activity in CD8; however, it has not been proven to be a reliable serum biomarker of tight control in Asian settings with different susceptibility genes and clinical characteristics from Western countries9. Moreover, another critical issue is inadequate data on optimal standards of biomarkers including serum CRP.The efficacy of the treat-to-target approach using biomarkers was assessed in a randomized controlled trial In this study, we sought to investigate whether tight control management based only on serum biomarkers that can be used in routine clinical practice leads to better outcome, and to identify their optimal standards for tight control management in Japanese patients with CD who were treated with anti-tumor necrosis factor (TNF) agents.10. Patients were included irrespective of the disease location or disease behavior. We excluded patients who discontinued treatment with anti-TNF agents for some reasons within 8 weeks. The Ethics Committee of the Tohoku University Hospital approved the study; the clinical procedures were carried out in accordance with the Declaration of Helsinki. Written informed consent was obtained from all patients.We reviewed the treatment course of consecutive patients with CD who were naïve to biologics and were treated with anti-TNF agents at the Tohoku University Hospital between July 2003 and September 2018. CD was diagnosed based on the diagnostic criteria proposed by the Research Group of Intractable Inflammatory Bowel Disease under the aegis of the Ministry of Health, Labor and Welfare of Japan11. Adalimumab was administered as 160 mg at week 0, 80 mg at week 2, 40 mg at week 4, and then continued at the same dose (40 mg) every 2 weeks. Patients who exhibited loss of response were managed by dose optimization of adalimumab (dose increase to 80 mg)11. In Japan, neither interval shortening of adalimumab to every week nor measurement of serum anti-TNF trough level are covered under the health insurance system.Patients were treated with anti-TNF agents . We administered 5 mg/kg of infliximab at weeks 0, 2, and 6, and then continued the same dose (5 mg/kg) every 8 weeks. Patients who exhibited loss of response were managed by dose optimization of infliximab We reviewed the results of blood investigations, and defined the indices of tight control based on the upper quartile of serum CRP and the lower quartile of serum albumin (Alb) at week 24. The achievement of tight control was judged at week 8 or 24 in each case.The primary outcome was major adverse outcomes . The association between failure to achieve tight control at week 8 or 24 and subsequent MAOs were analyzed. The secondary outcomes were clinical factors  and serum biomarkers (CRP and Alb at week 8 or 24) affecting the primary outcome.P values less than 0.05 were considered indicative of statistically significant difference.Data are presented as median value and interquartile range (IQR). Between-group differences were assessed using the Chi-squared test or Fisher’s exact probability test, as appropriate. The cumulative rate of MAOs was calculated using the Kaplan-Meier method, and between-group differences were assessed using the log-rank test. To identify factors affecting primary outcome, we also performed multivariate analyses using Cox proportional hazards model with clinical characteristics and serum biomarkers as covariates. The JMP Ver. 11 software  was used to perform all statistical analyses. A total of 245 patients were treated with anti-TNF agents . Of these, 22 patients were excluded because of relocation or surgery due to treatment failure within 8 weeks from the start of anti-TNF therapy. Thus, the treatment course was continued for more than 8 weeks by 223 patients; of these, 204 patients continued the treatment course for more than 24 weeks.Table At the start of anti-TNF therapy, median serum CRP and Alb levels were 0.6 mg/dL (IQR 25–75%: 0.1–2.1) and 3.7 g/dL (IQR 25–75%: 3.2–4.0), respectively. Infliximab was administered to 168 patients (75.3%), while 55 patients were administered adalimumab (24.7%). Owing to loss of response during the treatment course, 41 patients were managed by optimization of treatment: increase in dose of infliximab to 10 mg/kg in 20 patients; increase in dose of adalimumab to 80 mg in 5 patients; addition of prednisolone in 2 patients; addition of immunosuppressants in 14 patients (azathioprine in 12 patients and 6-mercaptopurine in 2 patients).During treatment with anti-TNF therapy, median serum CRP level improved to 0.1 mg/dL (IQR 25–75%: 0.1–0.2) and 0.1 mg/dL (IQR 25–75%: 0.1–0.4) at weeks 8 and 24, respectively. In the present study, index of tight control of CRP level corresponded to 0.4 mg/dL or less (<0.5 mg/dL) based on the upper quartile. Regarding CRP level at week 8, 188 patients (84.3%) achieved tight control, while 35 patients (15.7%) had CRP levels ≥ 0.5 mg/dL. Regarding CRP at week 24, 158 patients (77.5%) achieved tight control, while 46 patients (22.5%) had CRP levels ≥ 0.5 mg/dL.Similarly, median Alb level improved to 4.1 g/dL (IQR 25–75%: 3.8–4.4) and 4.1 g/dL (IQR 25–75%: 3.8–4.4) at weeks 8 and 24, respectively. Therefore, index of tight control in Alb level corresponded to ≥3.8 g/dL based on the lower quartile. Regarding Alb level at week 8, 169 patients (75.8%) achieved tight control, while 54 patients (24.2%) had Alb levels < 3.8 g/dL. Regarding Alb level at week 24, 159 patients (77.9%) achieved tight control, while 45 patients (22.1%) had Alb levels < 3.8 g/dL.Among the 223 patients who were followed up for >8 weeks , 88 patients experienced MAOs . Among the 204 patients who were followed up for >24 weeks , 80 patients experienced MAOs .On univariate analyses, the rates of MAOs were significantly higher in patients with penetrating type disease, CRP ≥ 0.5 mg/dL, and Alb < 3.8 g/dL at week 8 Table . We perfSimilar to the above results, the rates of MAOs were significantly higher in patients with penetrating type disease, CRP ≥ 0.5 mg/dL, and Alb < 3.8 g/dL at week 24 Table . On mult5 and the CALM study6, tight control management (treat-to-target strategy) of CD based on biomarkers has gained wide acceptance. Fecal calprotectin is now considered as the most reliable biomarker; however, it is not allowed in routine clinical practice in Asian and some Western countries. The present study suggests that tight control management may help avoid hospitalization, surgery, or discontinuation of anti-TNF therapy in Japanese CD patients with different susceptibility genes and clinical characteristics from Western countries. Even without monitoring fecal calprotectin, we may improve the disease course using only serum biomarkers (CRP and Alb) that can be used in routine clinical practice. Based on upper and lower quartiles, CRP < 0.5 mg/dL and Alb ≥ 3.8 g/dL were optimal standards for tight control management.Following the STRIDE guidelines8. However, CRP is a non-specific marker, and often increases in other infectious or inflammatory diseases unrelated to the gastrointestinal tract. In some studies, CRP levels were not associated with the disease activity in small intestinal lesions in patients with CD12. Despite the lack of strong correlation between CRP and intestinal inflammation, there are no alternative biomarkers for use in routine clinical practice. However, in the present study, CRP level at weeks 8 and 24 predicted the subsequent clinical course including hospitalization and surgery. In Asian settings, use of serum CRP alone may allow for tight control management of CD. In a study on Asian patients with quiescent CD, elevated CRP was found to be associated with poor outcomes13.Serum CRP has been used as a marker of disease activity in patients with CD6, CRP > 5 mg/L (CRP > 0.5 mg/dL) was used as one of the criteria for treatment failure in agreement with the present study.The optimal standard of CRP for management of disease activity is unclear. In the present study, optimal standard of CRP was set at <0.5 mg/dL based on the upper quartile, rather than at the normal reference level of <0.3 mg/dL for Japanese subjects. Results of multivariate analysis also suggested that CRP < 0.5 mg/dL was an effective biomarker of tight control. During multivariate analysis, inclusion of CRP < 0.3 mg/dL instead of CRP < 0.5 mg/dL did not generate a meaningful model. This may indicate that strict normalization of CRP is not required for tight control of disease activity. In the large multicenter CALM study15. This tendency was thought to be due to high clearance of anti-TNF agents in the presence of low serum albumin. The degree of clearance of anti-TNF agents cannot be proven in this study because measurement of serum anti-TNF trough level is not covered under the health insurance system in Japan; however, our results suggest that serum Alb as well as CRP may be an important target for tight control.Among serum biomarkers, Alb has also been shown to be associated with treatment course. Patients with low serum Alb at the start of anti-TNF therapy exhibited a tendency to relapse or require surgery earlier than that in patients with high serum albuminIn the present study, the optimal standard of serum Alb was set at ≥3.8 g/dL rather than at the normal reference level of ≥4.0 g/dL for Japanese subjects. On multivariate analysis, Alb ≥ 3.8 g/dL was also found to be an effective biomarker of tight control. During multivariate analysis, however, use of Alb ≥ 4.0 g/dL instead of Alb ≥ 3.8 g/dL did not identify any predictive factors. Although the extent of the lesion cannot be quantified, it might be difficult to perfectly normalize serum Alb level in patients with extensive disease or in those with a history of small intestinal resection. To predict the subsequent clinical course, strict normalization of serum Alb may not be required.17. Fecal calprotectin was shown to accurately mirror intestinal inflammation, and to correlate well with prognosis19. Unfortunately, it is not allowed in Asian and some Western countries. Our present study illustrates that it is possible to manage disease course with readily available blood tests. However, since the sensitivity and specificity of fecal calprotectin have been reported to be higher than CRP or other biomarkers20, a future prospective cohort study should include fecal calprotectin as one of the indices of tight control in Asian settings.In recent years, the efficacy of fecal calprotectin has been reported mainly in Western countries11. In general, most medical treatments are not very effective in patients with progressive disease including penetrating type disease. Inflammatory type disease, the so-called window of opportunity, might be the most suitable target for accelerated medical therapy22, although not actually proved in this study.In addition to serum and fecal biomarkers, disease behavior was associated with future disease course in the present study. Penetrating type disease has often been identified as a risk factor for poor prognosis24 were identified as risk factors for MAOs. In our gastroenterology division, the majority of patients are adults, while the percentage of younger patients (<17 years) is comparatively low. On the other hand, in Japan as well as in our gastroenterology division, the rate of CD patients with anal lesions is quite high26. These biased proportions may have influenced our results.We acknowledge several limitations in the present study. First, this was a single-center retrospective study. Second, neither age at diagnosis nor anal lesions which have often been found to be risk factors of poor prognosisIn conclusion, tight control management may help avoid MAOs. In Asian settings where fecal calprotectin is not allowed, CRP < 0.5 mg/dL and Alb ≥ 3.8 g/dL were the best candidate markers of tight control management. If we achieve tight control with optimization of anti-TNF therapy or additional immunomodulators, it might lead to improvement of disease course or better prognosis.


Neonatal hypertension is defined as persistent systolic and/or diastolic blood pressures above the 95th percentile compared to other infants of similar gestational age and size. Neonatal hypertension is a rare condition, occurring in only 0.2–3.0% of neonates. The most common etiology of neonatal hypertension is renal vascular or parenchymal disease, and it is usually detected on routine examination in an asymptomatic child. However, it may present in a variety of manners, including acute heart failure, renal dysfunction, feeding difficulties, failure to thrive, tachypnea, apnea, lethargy, irritability, or seizures.A term female was born via repeat caesarean section with vacuum extraction. On day of life (DOL) 3, the baby presented to the emergency department with poor feeding and lethargy. Initial laboratory tests indicated severe metabolic acidosis and the patient was transferred to our neonatal intensive care unit (NICU). During the hospital stay, the patient had intermittently high blood pressures. An echocardiogram was ordered, which demonstrated a severely decreased ejection fraction of 33%, but no signs of coarctation of the aorta. The low ejection fraction and constellation of symptoms were consistent with the diagnosis of acute heart failure, so treatment with milrinone was initiated. Further labs demonstrated elevated renin and aldosterone, and a computed tomography scan showed right kidney hypoplasia with reduced perfusion. This suggested a renovascular etiology of hypertension causing the initial presentation of acute heart failure. The patient was started on enalapril and clonidine for blood pressure control and was discharged with a home blood pressure monitoring system. At 5 months of life, this patient was still on enalapril and amlodipine as well as home blood pressure monitoring.Acute heart failure is a rare presentation of neonatal hypertension, and prompt recognition and treatment for the underlying systemic hypertension is necessary to provide the best possible outcomes for patients. Due to the lack of sufficient evidence, treatment of hypertension in newborns is often anecdotal in nature. Further awareness of neonatal hypertension and research determining ideal methods of diagnosis and treatment would benefit physicians and their affected patients. Neonatal hypertension is defined as persistent systolic and/or diastolic blood pressures above the 95th percentile compared to other infants of similar post-conceptual age, gestational age, and size . This coA term female was born to a 25-year-old gravida-2 para-1 at 39 + 1 weeks gestation. The mother had a history of gestational hypertension in the previous pregnancy, but blood pressures were within normal limits during this gestation. After an uneventful pregnancy, the patient was born via repeat caesarean section with vacuum extraction. The birth weight was 2.66 kg and APGARs were 8 and 9 at 1 and 5 min, respectively. In the postnatal period, the baby had mild respiratory distress, resolved with blow-by oxygen. Physiologic jaundice was treated with phototherapy and the patient was discharged on DOL 2.2 48/pO2 50/HCO3− 10.8/Base Deficit − 21. The patient was given sodium bicarbonate to resolve the acidosis and intubated prior to transfer. The patient was then transferred to a Level III NICU at our institution.On DOL 3, the baby presented to the same hospital with poor feeding and lethargy. Initial laboratory tests indicated severe metabolic acidosis with an arterial blood gas (ABG) pH 6.9/ pCO3/mcL, hemoglobin: 14.8 g/dL, platelet 288 × 103/mcL, neutrophils of 70% and lymphocytes of 18%, serum sodium:134 mmol/L, serum potassium: 6 mmol/L, serum bicarbonate: 15 mmol/L, serum blood urea nitrogen: 13 mg/dL, serum creatinine: 0.8 mg/dL, urine specific gravity: 1.004, serum lactic acid level: 7.4 mmol/L, which later worsened to 9.4 mmol/L and ammonia: 74mcmol/L. The patient’s blood pressure on admission was 119/70 mm of Hg in the right arm and 94/59 mm of Hg in the left arm. Empiric antibiotics  for suspected sepsis were initiated. However, cerebrospinal fluid studies, urinalysis, and blood cultures were later found to be negative, ruling out an infectious etiology.Upon admission, the differential diagnosis was inborn error of metabolism, shock due to sepsis and hypovolemia or cardiogenic etiology. Patient was kept nil my mouth (NPO), given intraveous fluids with dextrose10% water, and placed on mechanical ventilation. An high umbilical arterial catheter and umbilical venous catheter were placed in order to accurately monitor arterial and venous pressures, and obtain blood gases. Initial labs demonstrated white blood cells: 20.6 × 10The patient had intermittently high blood pressures during the hospital stay with systolic pressures in the 100’s mmHg, and diastolic pressures in the 70’s. An echocardiogram demonstrated mitral regurgitation, tricuspid regurgitation, and severely depressed left ventricular function with an ejection fraction of 33%. It also ruled out other structural cardiac anomalies, including coarctation of the aorta. The low ejection fraction and constellation of symptoms were consistent with the diagnosis of acute heart failure, so treatment with milrinone (1 mcg/kg/min) was initiated and the ejection fraction improved to 44%. The patient showed signs of improvement by DOL 5 with an ABG of 7.48/33.8/60/25/2 and was weaned off of mechanical ventilation. Trophic feeds were initiated; however this resulted in the patient becoming slightly acidotic with a pH of 7.32, so she was again made (NPO).The patient was transferred to a Level IV NICU with a metabolic specialist, where complete work up demonstrated no inborn errors of metabolism. A CT scan showed right kidney hypoplasia with reduced perfusion, and further labs demonstrated an elevated renin of 194 ng/ml/hr. Ref 2.0–35) and aldosterone of 1476 ng/dL (Ref 6.0–179.0). This suggested a renovascular etiology of hypertension causing the initial presentation of acute heart failure. After discharge, upon follow-up with the primary care physician (PCP), she was again found to have elevated blood pressures (data not available). The patient was admitted to another hospital and started on enalapril (1 mg/mL) and clonidine (0.1 mg/mL) for blood pressure control. The patient was discharged on this regimen with a home blood pressure monitoring system. She continued to have uncontrolled blood pressures (data not available) and was admitted again to our institution where amlodipine 0.1 mg/kg BID was added to her regimen, and adequate control was achieved. Repeat work up for pheochromocytoma with Urine 24 h vanillylmandelic acid was 0.5 mg/24 h, Urine 24 h normetanephrines 127 mcg/24 h, Urine 24 h metanephrine 12 mcg/24 h, were with in normal for age. Renal ultrasound: mild left renal pelviectasis. Otherwise, normal echogenicity of bilateral kidneys, Rt kidney was 3.9 × 2.3 × 2.7 cm and Left Kidney 5.1 × 2.5 × 2.9 cm  inhibitors, calcium channel blockers, beta blockers, and alpha antagonists . ACE inhThe prognosis for infants with hypertension depends on the underlying cause. The majority of cases resolve, and only 15% of patients continue to require medications by 6 months of age . ImproviThis is an unusual case of hypertension in a newborn presenting as acute heart failure. Prompt recognition of systemic hypertension as the cause of systolic dysfunction is necessary to facilitate early treatment and prevention of further complications of severely elevated blood pressures. In addition to furthering awareness of this uncommon neonatal condition and various presentations, we assert that there is great need for further research to support efficacy, safety, and standardization of therapeutic interventions for neonatal hypertension.


We report a case of nivolumab‐induced delayed‐onset aseptic meningitis and a case of limbic encephalitis and peripheral nerve palsy with toxicity relapse 6 months after initial presentation. The atypical presentations contribute to our knowledge of these rare events and reinforce the necessity for vigilant monitoring and a multidisciplinary treatment approach. We report a case of nivolumab‐induced delayed‐onset aseptic meningitis and a case of limbic encephalitis and peripheral nerve palsy with toxicity relapse 6 months after initial presentation. The atypical presentations contribute to our knowledge of these rare events and reinforce the necessity for vigilant monitoring and a multidisciplinary treatment approach. Here, we review the literature and report two cases, a rare case of nivolumab‐induced delayed‐onset aseptic meningitis and a case of limbic encephalitis and peripheral nerve palsy with toxicity relapse 6 months after initial presentation.Within the past decade, immune checkpoint inhibitors (ICIs) have demonstrated survival advantages in various solid tumors and are now a therapeutic pillar in oncology. The primary function of immune checkpoints is to maintain immune homeostasis by down‐regulating T‐cell activation.Since 2011, 7 ICIs have been approved by the US Food and Drug Administration: ipilimumab, an inhibitor of cytotoxic T‐lymphocyte‐associated protein 4 (CTLA‐4); nivolumab, pembrolizumab, and cemiplimab‐rwlc, which inhibit programmed cell death protein 1 (PD‐1); and atezolizumab, avelumab, and durvalumab, which inhibit PD‐L1. Urothelial carcinoma (UC) is one of the many tumors that has demonstrated response to ICIs. Five PD‐1/PD‐L1 inhibitors have produced meaningful response rates in platinum‐refractory UC.Although robust data support the use of immunotherapies in many solid tumors, including UC, the efficacy of these agents in renal medullary carcinoma (RMC) is not well established. Published case reports provide a hint of activity in this rare disease.Severe immune‐related adverse events (irAEs), defined as grade ≥3, are estimated to occur in approximately 22%, 7.1%, and 6.3% of patients receiving a CTLA‐4, PD‐1, or PD‐L1 inhibitor, respectively.22.1A 58‐year‐old male with UC metastatic to the lung and lymph nodes who initially presented with 5 years of intermittent hematuria was found to have a left renal pelvis mass status postleft nephroureterectomy for a pT3Nx high‐grade UC followed by 4 cycles of adjuvant gemcitabine plus cisplatin with metastatic recurrence 13 months later. He was treated with 3 cycles of gemcitabine plus carboplatin without response. Relevant past medical history included obesity, noninsulin‐dependent diabetes mellitus, hypertension, hyperlipidemia, fatty liver disease, anxiety, depression, and obstructive sleep apnea. Following enrollment on a phase I clinical trial (NCT02496208), treatment was initiated with cabozantinib 40 mg p.o. once daily plus iv nivolumab 1 mg/kg every 2 weeks.The first 12 cycles (48 weeks) of treatment were unremarkable. The patient achieved a complete response in the hilar and retrocrural lymph nodes and a partial response (PR) in the lung by cycle 11. Restaging evaluations at this time revealed a continuing PR to treatment, with a 51% reduction in the target right lower lobe mass and lymph node lesions. Soon thereafter, the patient presented with a fever of 39.3°C, chills, malaise, dry cough, headache, and bilateral eye pain. The patient described a dull frontal headache not relieved by analgesics and pain with ocular movement, but a neurologic examination was otherwise unremarkable. Concomitant medications were unchanged and included oxycodone, citalopram, loperamide, metoprolol succinate, opium tincture, rosuvastatin, aspirin, cholecalciferol, fexofenadine, levothyroxine, sitagliptin, and lisinopril. Treatment with nivolumab and cabozantinib was held. Three days later, the patient complained of worsening ocular pain with movement and intermittent shooting, lancinating right ear pain. MRI revealed normal orbits and a punctate focus of leptomeningeal enhancement  of therapy. A biopsy of a residual subcutaneous nodule revealed necrotic tissue. Treatment was complicated with bilateral anterior uveitis that presented as blurry vision and resolved with topical agents. A short time later, the patient presented with a 5‐day history of diplopia, headache, photophobia, and difficulty with short‐term memory. Neurologic examination was significant for impaired delayed recall, right 6th cranial nerve palsy, and ataxia. MRI revealed a signal abnormality in bilateral mesial temporal lobes Figure . FindingThe patient completed the steroid taper and was not rechallenged with immunotherapy. She remained neurologically stable until approximately 6 months after the initial presentation, at which time she presented with a 2‐week history of confusion, paranoia, and short‐term memory impairment. Neurologic examination revealed symptoms of contralateral left 6th cranial nerve palsy and a Montreal Cognitive Assessment of 11/30. MRI was notable for progression of atrophy but no alteration or enhancement to account for the new‐onset palsy. Cerebrospinal fluid (CSF) analysis is provided in Table 3We present 2 cases of serious neurologic irAEs, aseptic meningitis and limbic encephalitis, both associated with the use of ICIs. Both patients had remarkable durable disease responses to immunotherapy. The patient with UC experienced a durable (approximately 2 years) PR on the nivolumab/cabozantinib regimen. Similarly, the patient with RMC experienced a PR on the nivolumab/ipilimumab/cabozantinib combination for approximately 5 months. Emerging data have begun to establish a correlation between irAEs and the efficacy of ICIs in patients with solid tumorsDrug‐induced aseptic meningitis is a rare adverse event that has been reported with various pharmaceutical products, including nonsteroidal anti‐inflammatory drugs, antimicrobials, iv immunoglobulin (IVIG), vaccines,Patient 2 met the diagnostic criteria for autoimmune limbic encephalitisOn brain autopsy, sections of patient 2's cerebellum showed a marked loss of Purkinje cells accompanied by microglial activation and Bergman gliosis. The cerebellum is a frequent target of paraneoplastic autoimmunity. With paraneoplastic cerebellar degeneration (PCD), pathologic examination shows marked degeneration of Purkinje cells with minimal involvement of the molecular or granular cell layers. Depending on disease stage, inflammation can be marked, sparse, or absent in the cerebellar cortex.Although reports of aseptic meningitis and limbic encephalitis after treatment with ICIs have previously been published, these cases help expand our knowledge of these rare but potentially serious neurologic toxicities. Patient 1 provides evidence of an unusual late‐onset aseptic meningitis following 24 doses of nivolumab. Patient 2 is a unique case of recurrent limbic encephalitis. Neurologic sequelae from these agents may occur, requiring prompt attention and a multidisciplinary approach to reduce morbidity and mortality.None declared.LMC, NND, and ABA: made major contributions to the writing of this manuscript. All authors read and approved the final manuscript.


Clinical characteristics combined with parenchymal volume did not improve the equation over parenchymal volume alone. These results support the feasibility of estimating nephron number by a combination of unenhanced CT and biopsy-based stereology, with a possible application for renal disease patients who are often not suitable for contrast media.Methods for estimating nephron number in a clinical setting may be useful for predicting renal outcomes. This study aimed to establish such a method using unenhanced computed tomography (CT) and biopsy-based stereology. Patients or living kidney donors simultaneously subjected to enhanced and unenhanced CT examinations were randomly assigned to development and validation groups. The enhanced CT-measured arterial phase and the venous phase images of kidneys were regarded as the true values for cortical volume and parenchymal volume, respectively. Linear multiple regression analysis was used to create models for estimating cortical volume using explanatory variables including unenhanced CT-measured parenchymal volume. Nephron number was determined as the product of cortical volume and the glomerular density in biopsies of donors. Five equations for estimating cortical volume were created and verified. In donors, estimated nephron number by unenhanced CT was consistent with that by enhanced CT, with minimal errors in all models (636–655 ± 210–219 vs. 648 ± 224 × 10 Recent studies have reported a method for measuring cortical volume using images of contrast-enhanced computed tomography (CT)8. With this method, the intensified part of the arterial phase following injection of contrast medium is regarded as cortical volume. By combining enhanced CT-measured cortical volume and glomerular numerical density obtained using stereological analysis of implantation biopsies from American living kidney donors, a method for estimating total nephron number per kidney was proposed9. The findings were consistent with those in previously reported autopsy series of American Caucasians obtained using the disector/fractionator method, the current gold-standard method for estimating total nephron number10. We recently used a combined CT angiography and biopsy-based approach to estimate nephron number in Japanese living kidney donors11. The estimated total nephron number obtained in the study was quite similar to that reported in a previous Japanese autopsy study using the disector/fractionator method, again confirming feasibility of the method12.Renal cortical volume has previously been used as a surrogate measure of renal functionDespite the potential utility of estimating cortical volume using CT angiography in the clinical setting, the method has a serious disadvantage in that the contrast media is not preferable for the majority of patients with renal disease and/or renal impairment. In the present study, we therefore aimed to create a regression equation for estimating cortical volume using unenhanced CT-measured parenchymal volume, which can be applied to renal disease patients. In addition, we validated the method for estimating nephron number by combining an equation-based cortical volume with biopsy-based stereology.This study included 49 kidney donors and 58 subjects with renal disease. Clinical characteristics of the subjects are provided in Table 3 per kidney and that based on unenhanced CT was 127.2 ± 31.0 cm3 per kidney. As shown in Fig. The value of the cortical volume based on enhanced CT was 126.3 ± 30.1 cm3 per kidney. In univariate analyses, neither diabetes nor hypertension were significantly associated with cortical volume, and thus were excluded from the models. Based on multivariate analyses, we created four models by using sequential regression to estimate cortical volume as follows : (i) Equation 1, estimated CV (cm3) = −17.1 (intercept) + 0.15 × eGFR (mL/min/1.73 m2) + 0.076 × body height (cm) + 0.67 × PV (cm3); (ii) Equation 2, estimated CV (cm3) = 35.4 (intercept) − 0.2 × age (year) + 4.77  − 13.2 × log [Cr (mg/dL)] − 0.14 × body height (cm) + 0.67 × PV (cm3); (iii) Equation 3, estimated CV (cm3) = −15.5 (intercept) + 0.17 × eGFR (mL/min/1.73 m2) + 0.25 × body weight (kg) + 0.62 × PV (cm3); (iv) Equation 4, estimated CV (cm3) = 0.4 (intercept) − 0.12 × age (year) + 1.29  − 15.8 × log[Cr (mg/dL)] + 0.23 × body weight (kg) + 0.63 × PV (cm3). By adding all these variables and using the stepwise selection method with Akaike information criteria, the same equation as Equation 3 was created. We also created a regression equation of ‘model 0’ using only PV as an explanatory variable: Equation 0, estimated CV (cm3) = −1.3 (intercept) + 0.71 × PV (cm3). In each regression equation, the estimated cortical volume showed strong correlations with measured cortical volume with small root mean squared error (RMSE) and mean absolute error (MAE), regardless of whether they were donors or diseased patients as shown in Table 3The univariate and multivariate analyses of factors associated with cortical volume estimates obtained using enhanced CT images are shown in Table We performed additional analyses to confirm that the severity of renal risk factors, including diabetes and hypertension, were not associated with CV. Two-way ANOVA between CKD category in relation to diabetes or hypertension was performed to examine whether there was any influence on CV depending on the stage of CKD . In each regression equation, the estimated CV showed significant correlations with measured CV, with small RMSE and MAE . Five cases were excluded due to insufficient numbers of glomeruli in the biopsies. The non-sclerotic glomerular density and the total glomerular density were 13.5 ± 4.7/mmThe aim of this study was to produce a model to estimate renal cortical volume using parenchymal volume measured using unenhanced CT imaging. Body size (body weight or body height), renal function (eGFR or Cr), diabetes, hypertension, age, gender, and parenchymal volume were selected as candidate variables for the regression equation models. In univariate analyses, neither diabetes nor hypertension were significantly associated with cortical volume and were therefore excluded from explanatory variables. Using multivariate analyses, we produced four regression equation models for the estimation of cortical volume using unenhanced CT images. We also created a simple model with only parenchymal volume as an explanatory variable for comparison. The cortical volume values obtained by the models showed strong correlations with the measured cortical volumes obtained using the enhanced CT-based method, for both kidney donors and patients with disease. In addition, we confirmed the validity of the models in a validation group. These results suggest that the regression equations produced in this study can produce accurate and precise estimates of cortical volume.3 per kidney. This value is consistent with, although somewhat lower than previous reports of cortical volume of approximately 90 to 120 cm3 per kidney7. Previous studies have shown that a range of factors influence cortical volume including age, gender, renal function, diabetes, hypertension, body size, and renal morphological parameters14. In this study, we tried to produce a model by applying these factors as candidate explanatory variables for cortical volume. First, we selected Cr as a renal function parameter. We additionally created a model based on eGFR without age, gender or Cr because eGFR is a renal function parameter calculated from these parameters, and age-related decreases in cortical volume have been reported to correlate with GFR5. In fact, in the present study, no single component of eGFR  was more strongly correlated with the enhanced CT-measured cortical volume than eGFR itself in univariate regression analyses. We therefore concluded that eGFR is applicable to one of the models for estimating cortical volume. Second, both body height and body weight are reported as factors related to cortical volume3. The present findings confirmed these relationships. Third, renal morphological metrics such as renal thickness, width and length have been examined as explanatory factors for cortical volume in previous studies15. In this study, parenchymal volume was used as an explanatory variable, since we considered it may be morphologically more homologous to cortical volume than thickness and length measurements. Our results clearly demonstrated that parenchymal volume was the strongest variable in the determination of cortical volume, though other variables such as body size and renal function metrics were also identified as significant determinants of cortical volume.In this study, the mean value for measured cortical volume was 89.3 cm3 (within 2% to the cortex) among the models, suggesting that this difference can be clinically ignored. These results suggest that clinical characteristics combined with parenchymal volume did not meaningfully improve the equation over parenchymal volume alone. This is probably due to the fact that parenchymal volume had the strongest influence in each model.We hypothesized that clinical characteristics could be combined with parenchymal volume to better estimate cortical volume. We therefore produced five variations of the regression model based on age, gender, renal function, body size and parenchymal volume. Although the equation based on age, gender, log [Cr], body weight and parenchymal volume was statistically the most suitable model of the five equations, the validity of each model was confirmed. In the validation group, the equation that used only parenchymal volume had the highest fitness and the lowest value of Akaike information criteria. However, the differences in RMSE and MAE were up to 1.65 and 1.32 cmIn clinical settings, body weight can change depending on the etiology of the renal disease, especially in such cases as nephrotic syndrome with systemic edema. Such variability could influence an estimate of cortical volume if body weight was included in an equation model. Thus, the body size parameters in these equation models should be modified depending on the type of renal disease, and possibly a model using body height or a model that uses only parenchymal volume would be more appropriate in some cases.17. Similar compensatory changes may occur in response to nephron loss in patients with chronic renal diseases18. Thus, there remains an issue of difficulty to interpret the number of nephrons in an individual based solely on knowledge of cortical volume. In fact, previous reports of cortical volume did not consistently find correlations with either nephron number or renal outcomes20.Although the number of nephrons does not increase in humans after term birth, each nephron has the potential to undergo hypertrophy and thus contribute more to renal function. In subjects with low nephron number, enlargement of the renal cortex due to compensatory hypertrophy of individual nephrons occurs in association with increasing demands such as normal body growth and catch-up growth22. Although differences in nephron number have been extensively investigated in different racial and socioeconomic groups, until recently, data for Asian populations including Japanese subjects was lacking. Recently, we estimated nephron number in Japanese subjects using the gold-standard disector/fractionator method and reported that values were significantly lower than in other races12. Estimating the number of individual nephrons is considered to be clinically useful for assessing the future progression of kidney diseases. However, the disector/fractionator method, which is the standard method for accurately measuring the total number of nephrons, requires a great deal of labor and time and cannot be applied to living subjects23. In the present study, we confirmed that the difference between enhanced or unenhanced CT-based methods for estimating nephron number was minimal. Interestingly, greater variation was found in estimates of subject glomerular density than cortical volume. Thus, our results suggest that the unenhanced CT method can be used to estimate cortical volume as well as nephron number. This approach may therefore allow us to estimate nephron number in renal disease patients who are often not suitable for contrast-enhanced CT imaging.Differences in nephron number may also be indicative of differences in renal functional reserve between individuals with similar clinical characteristics at the time of diagnosis of kidney disease, which is recognized as one cause of divergent renal outcomes4. This is the “trade-off” with some modest loss of accuracy but avoiding contrast nephropathy. Further, our equation may not be applicable in advanced CKD, where much more severely disproportionate loss of CV  occurs. In addition, the results of this study need further study in children and in other races than Japanese adult subjects. However, we expect that with minor modifications this methodology would be applicable to other subjects.Limitations of this study include the relatively small number of patients with renal impairment since nearly half of the subjects were living kidney donors. Patients with heavy proteinuria tend to be avoided for using contrast agents and may not be included in our subjects. While PV can be used to estimate CV, PV is an imperfect surrogate for CV as some clinical factors associate with CV differently than PVIn conclusion, this study provides regression equation models to estimate renal cortical volume by using unenhanced CT images. Estimation of cortical volume by this method may be useful for the estimation of individual nephron number as well as prediction of renal outcomes, with a possible application for patients with renal disease.24. 107 subjects including 49 donors and 58 patients were recruited, and they were randomly assigned into the derivation group (N = 80) for model creation and the validation group (N = 27). This study was approved by the ethics review board of the Jikei University School of Medicine [30–229 (9250)]. Since this study used only existing clinical data, we provided participants with information about the opportunity to opt out and obtained a waiver of informed consent.In this study, we reviewed images and clinical data from diseased patients and living kidney donors in our department from 2007 to 2017 who underwent abdominal contrast-enhanced CT, in which whole kidney images were included. All patients were simultaneously subjected to unenhanced and contrast-enhanced CT examinations at the same time. Kidney donors were selected according to the Amsterdam Forum guidelines11 using software  to semi-automatically segment the cortex and medulla from transverse images obtained during the arterial phase of the CT angiogram , the Definition AS+  or the Definition flash . The thickness of the obtained image was in the range of 3.0 to 5.0 mm. The arterial phase and venous phase images were obtained 30 and 90 seconds after infusion of intravenous contrast, respectively. Renal cortical volumes were measured as previously describedram Fig. . Parenchram Fig.  and the ram Fig. .Figure 3et al.9. Donors with less than five glomeruli in their biopsy were excluded from the analysis of nephron number. Nephron number was defined as the number of non-sclerotic glomeruli (NSG) and was calculated by multiplying total cortical volume (mm3) by the numerical density of NSG (number per mm3 of cortex)  Fig. . These vIn biopsies, the combined areas of all glomerular capillary tufts and the area of renal cortex were measured using a computerized image analyzer . Glomerular tuft area was defined as the area of the outer capillary loops of the tuft Fig. . Glomeru3 of cortex) was calculated using the Weibel and Gomez stereological method as follows25: 3 of cortex) was also calculated: 3 of cortex) was the sum of NSG density and sclerotic glomerular density. We previously reported nephron number estimated by enhanced CT-based methods in the same donor group11.The total number of NSG was obtained by summing the numbers of complete and partial NSG (counted as 0.5 NSG). The mean area of NSG per biopsy was obtained by dividing the total area of all NSG by the total number of NSG. NSG density 26. Chronic kidney disease (CKD) stages were defined as follows: G1, GFR ≥ 90 mL/min/1.73 m2; G2, GFR 60–89 mL/min/1.73 m2; G3, eGFR 30–59 mL/min/1.73 m2; G4, 15–29 mL/min/1.73 m2; G5, GFR < 15 mL/min/1.73 m2. Elderly was defined as 60 years and older.Blood pressure was measured with the subject resting in a sitting position. Serum creatinine (Cr) was measured using the enzymatic method. These measured values, together with other blood laboratory values, body height and body weight were the latest available data before CT imaging. Hypertension was defined as a systolic blood pressure of >140 mmHg and/or diastolic blood pressure of >90 mmHg, or the use of antihypertensive medications. The estimated glomerular filtration rate (eGFR) was calculated from serum Cr using a modified equation for the GFR of Japanese individuals: eGFR = 194 × age2 test for categorical variables were used.Continuous variables were assessed for normality both visually  and by inferential statistics (Shapiro–Wilk tests) and are presented as mean ± standard deviation. Serum Cr was shown as median (interquartile range) in the clinical characteristics and was naturally log-transformed in the regression analysis due to the skewed distributions. Categorical variables were summarized by the frequency. For comparisons between the derivation group and the validation group, an unpaired t-test for continuous variables, a Mann-Whitney test for serum Cr, and a χ27. Other R packages were used for analyses as follows: (i) ‘Metrics’ package for calculating the RMSE and the MAE, (ii) ‘ggplot2’ package for drawing scatter plots and regression lines.Correlations were assessed using Pearson’s correlation coefficient. Previously reported or clinically relevant parameters were subjected to univariate or multivariate linear regression analyses in order to determine the model of estimated cortical volume. In addition to age, gender and renal function, height and weight were adopted as body size factors as an explanatory variable reported to be related to cortical volume. The absolute bias, which is the mean differences between the measured cortical volume and the estimated cortical volume, was calculated. The relative bias, which is the division of the mean differences between the measured cortical volume and the estimated cortical volume, was calculated. Two-sided P values of <0.05 were considered statistically significant. All statistical analyses were performed using EZR , which is based on the R software package This study was approved by the ethics review board of the Jikei University School of Medicine [30-229 (9250)]. Since this study used only existing clinical data, we obtained a waiver of informed consent in accordance with relevant guidelines and regulations.Supplementary information


Bioelectromagnetics Published by Wiley Periodicals, Inc..Melanocytes in hair are located around dermal papilla cells at the tip of the hair follicle. In this study, we examined the melanogenesis of a three‐dimensional (3D) hair dermal papilla model treated with natural extracts and electromagnetic fields (EMFs). The 3D model involved dermal papilla‐like tissue (DPLT), an aggregation of a mixture of dermal papilla cells, and melanocytes in microwells. Rice bran extract (RBE), an EMF, and RBE/EMF were applied to different DPLT groups. The LDH assay indicated no cell stress in all experimental groups, and detection of tyrosinase activity demonstrated high activity in the RBE/EMF group. Western blot analysis of the RBE, EMF, and RBE/EMF groups revealed increased MITF, TRP‐1, and tyrosinase expression. In addition, the mRNA expression of ET‐1, laminin, bFGF, β‐catenin, MITF, and tyrosinase was increased in the RBE/EMF group, as demonstrated by RT‐qPCR analysis. HMB45 and Fontana–Masson immunostaining showed that the RBE/EMF group had the highest melanin content. Therefore, RBE and EMF may be used as a material and therapy, respectively, for the treatment of vitiligo and white hair, through activation of melanogenesis in melanocytes. Bioelectromagnetics. 39:595–603, 2018. © 2018 The Authors.  In the human scalp, hair follicles display cyclical activity with each cycle being characterized by three successive phases  that have distinct durations [Commo and Bernard, Ardisia crenata, scoparone, mangosteen leaf, and Kaliziri [Cho et al., In hair, melanin is a compound that determines hair color. Gray hair is affected not only by genetic factors but also by strong ultraviolet (UV) light and stress. Melanogenesis is a physiological process resulting in the production of melanin, which plays an important role in the prevention of sun‐induced skin injury and contributes to skin and hair color [Cho et al., It is widely known that extremely low‐frequency electromagnetic fields (ELF‐EMFs) can interact with biological systems and affect health [Cho et al., Pigment production and deposition are restricted to the hair follicle and hair shaft, respectively. During the active growth phase (anagen) of the mature hair follicle, pigments are synthesized by melanocytes in the hair bulb at the base of the follicle and adjacent to the dermal papilla, a specialized mesenchymal component of the hair follicle that plays important roles in controlling follicle morphogenesis, stem cell activity, hair shaft formation, and pigmentation [Enshell‐Seijffers et al., We prepared DPLT as 3D aggregates similar to those under in vivo conditions, and a hair model was produced by adhering melanocytes to the DPLT. The effects of RBE and EMFs on melanogenesis were examined, and the synergistic effects of simultaneous stimulation with RBE and EMF were evaluated. After DPLT culture, tyrosinase activity and lactate dehydrogenase (LDH) were analyzed, and western blot analyses of MITF, TRP‐1, tyrosinase, and p‐CREB were performed. Furthermore, the mRNAs of endothelin‐1 (ET‐1), laminin, basic fibroblast growth factor (bFGF), β‐catenin, MITF, and tyrosinase were analyzed, and HMB45 and Fontana–Masson immunostainings were assessed.RBE was produced using the method described by Jang and Seo . RBE was2. The culture medium was changed once every 2 ∼ 3 days, and the cells were subcultured several times until a sufficient number of cells was obtained.This study was approved by the Institutional Review Board of Dongguk University . Human hair dermal papilla cells were used separately from donated hair follicles. The culture medium was prepared by adding 10% fetal bovine serum (FBS) to high glucose Dulbecco–Vogt modified Eagle's minimal essential medium (DMEM) and placed in a humidified incubator at 37 °C in 5% CO2. The culture medium was changed every 2–3 days, and the cells were passaged 3–7 times. To induce differentiation, melanoblasts were exposed to melanocyte differentiation induction medium, consisting of M254, HMGS‐2, 10 nM alpha‐melanocyte stimulating hormone , 10 nM 12‐O‐tetradecanoyl‐phorbol‐13‐acetate , and 20 μM forskolin . After 3 days, the differentiation induction medium was replaced with M254 before the cells were used.Melanocyte differentiation was carried out under specific culture conditions based on the method of Cho et al. . Melanob5 dermal papilla cells were seeded in each concave microwell and cultured with high glucose DMEM + 10% FBS. After dermal papilla cells were seeded for 3 days, 5 × 105 differentiated melanocytes were seeded in each concave microwell containing the dermal papilla spheroids. At this time, the medium was replaced with M254 by removing high glucose DMEM + 10% FBS, and the experiment was conducted 3 days after the melanocytes were seeded.DPLT spheroids were formed in silicon elastomer‐based concave microwells  with 400 μm diameters. Subsequently, 2.5 × 10A total of five DPLT spheroid groups were performed. As controls, a negative control group cultured in M254 and a positive control group cultured in M254 supplemented with 10 mM α‐MSH were used. The experimental groups were divided into three groups: DPLT spheroids treated with RBE (30 μl/ml), EMF, and RBE/EMF.2 and operated at a magnetic field of 0.2 mT, and a frequency of 60 Hz, for 30 min/day for 3 days . The medium (100 μl) was placed in a 96‐well plate with 50 μl of working solution and incubated at room temperature for 30 min. Then, the reaction was terminated with 50 μl of stop solution (1 N HCl), and the absorbance was measured at 570 nm.Tyrosinase activity was measured, based on a method described by Jang and Seo . The DPLAfter culture, the DPLT was lysed, using a buffer containing 2% SDS, 0.1 mg/ml bromophenol blue in Tris‐HCl (pH 6.8), 5% 2‐mercaptoethanol, and 10% glycerol, and boiled at 100 °C for 10 min. The protein was quantified by BCA . Subsequently, the sample was subjected to SDS‐polyacrylamide gel electrophoresis, and the protein was transferred from the gel to a nitrocellulose membrane. The primary antibodies used were β‐actin , TRP‐1, tyrosinase, MITF, and p‐CREB . Blotting images were obtained by the Molecular Imager ChemiDoc XRS+ system with Lumi Femto . The Western blot images were quantified using ImageJ software .The overall reverse transcription quantitative real‐time polymerase chain reaction (RT‐qPCR) procedure was based on the experimental method detailed by Cho et al. . The tot2O2. The nonspecific reaction of bovine serum albumin and non‐immune serum was conducted with the respective monoclonal antibody of anti‐HMB45  following standard immunohistochemical procedures or with anti‐goat or anti‐rabbit immunoglobulin using the avidin–biotin–peroxidase complex (ABC) method. Either aminoethyl carbazole (AEC) or 3,3′‐diaminobenzidine (DAB) was used as the substrate.Immunohistochemical analysis was conducted based on methods described by Seo et al.  and Kim Fontana–Masson silver staining was performed using a previously described method with formalin‐fixed samples stained with silver nitrate  for 1 h at 56 °C, followed by washing with distilled water. Then, the samples were fixed in 5% sodium thiosulfate solution  for 5 min and washed with distilled water. Next, they were stained with nuclear fast red solution  for 5 min and washed three times with distilled water. After dehydration with 95% ethanol and 100% ethanol, the samples were washed two times with xylene . The F/M and HMB45 staining area densities were normalized using ImageJ software.t‐test. Difference between means were considered significant when P < 0.05 . Graphical representations were produced with SigmaPlot .Results are reported as the mean ± standard error (SE), and each experiment was repeated at least three times. Data were analyzed by the Student's Figure The efficacy of RBE and EMF was evaluated using DPLT with a negative control, a positive control, RBE 30 μl/ml), EMF , and RBE/EMF . After 3 days of substance administration and irradiation, the medium in which the cells were cultured was collected to measure the amount of LDH. The LDH assay revealed that the number of cells was similar to or lower than that of the negative control group, and no specific cell damage was observed in the RBE/EMF group  are tightly regulated by MITF [Tuerxuntayi et al., The dermal papilla cells produce and secrete molecules and growth factors that make up the extracellular matrix (ECM) and cytokines, such as bFGF, ET‐1, and SCF [Lu et al., Additionally, the ECM component of cultured human dermal papilla cells stimulates the tyrosinase activity of melanocytes [Buffey et al., We found that the gene expression of dermal papilla cells and melanocytes was increased when DPLT was treated with RBE and EMF. Notably, the overall mRNA and protein expression level in the experimental group treated with both RBE and EMF significantly increased. The increased expression of ET‐1 and bFGF, induced by increasing p‐CREB expression in the group simultaneously administered RBE and EMF, increased melanogenesis.We demonstrated that the melanocytes of the DPLT were activated by RBE, EMF, and RBE/EMF. RBE, EMF, and RBE/EMF induced the p‐CREB signal, and this signal activated TRP‐1, tyrosinase, and MITF, and increased melanogenesis. Notably, simultaneous stimulation with RBE and EMF had a synergistic effect on increasing melanogenesis. The results of our study can be used as a reference for applying RBE/EMF to a human model. Furthermore, RBE and EMF may be used in the future as a material and therapy, respectively, for the treatment of vitiligo and white hair, through activation of melanogenesis in melanocytes.