Risk factors associated with opioid-induced adverse reactions (OIARs) in the elderly population have not been well defined. The objective of this study was to determine effects of various risk factors on incidence of OIARs in male elderly patients.A retrospective cohort study in Korea Veterans Hospital was performed. Data were analyzed in male patients aged 65 years and older who received morphine, oxycodone, or codeine. Binomial variables describing patient-related and drug-related characteristics were constructed. Associations between these variables and frequency of OIARs were determined. Odds ratio (OR) and adjusted odds ratio (AOR) were calculated from univariate and multivariable analyses, respectively. Attributable risk was obtained by (1–1/OR)*100%.n = 59) and central nerve system adverse effects (n = 20). The odds of OIARs in patients with opioid use ≥12 weeks was increased by 80% compared to those with opioid use < 12 weeks. Attributable risk of GABA analogues was 64~78% in constructed Models. Compared to codeine users, patients using morphine and oxycodone had 653 and 473% increased odds for OIARs, respectively. MME ≥ 60 mg/day had a 317% increased odds for OIARs (95% CI: 1.92–9.04) compared to MME < 60 mg/day. Opioid combination therapy had a 139% increased odds for OIARs compared to monotherapy.Of 316 patients, 28% experienced at least one adverse event. The most common adverse events were gastrointestinal problems (These findings have significant implications for clinical use of opioid in elderly patients. Our study suggests that low dose short-term use will pose less risk of OIARs for the elderly, whereas concomitant use of GABA analogues, strong opioids and dual-opioid therapy may increase the risk of OIARs. Therefore, clinician should carefully monitor patients when starting opioid therapy in older population. Over the past 20 years, therapeutic use of opioids for both cancer and non-cancer pain has increased dramatically in developed countries , 2. In mIn parallel with increased opioid dosages and patient access to opioids, there is an increasing concern about their adverse effects among health care providers, in addition to misuse and addiction problems. Opioid-induced adverse reactions (OIARs) are well-known. They may not differ between younger adults and older adults. However, some OIARs stemming from drug interactions are more specific to older adults considering their high incidence of polypharmacy . More prThe extent and nature of OIARs depend on many factors. Previous studies have assessed the association between patient-factors  and incidence of some OIARs with short-term use in adults . HoweverTherefore, the objective of this cohort study was to focus on the elderly population and investigate patient-related and drug-related risk factors for OIARs with short-term and long-term use. The association between respective risk factors and commonly encountered OIARs such as gastrointestinal (GI) and central nerve system (CNS) adverse reactions was also analyzed.This study is a retrospective study which was conducted at Korea Veterans Hospital, a 1400-bed hospital in Seoul, between January 2012 and December 2012. In this hospital, people ≥65 years of age comprise the majority (about 70% of over 4000 patients per year) of the patients and opioid initiations are common among veterans with chronic pain. All ambulatory patients with the following characteristics were included: 1) was administered with a single component oral opioid  for at least a day between January 1, 2012 and December 31, 2012, 2) were at least 65 years of age when their first opioid was prescribed. In our study period, opioids were prescribed for several clinical symptoms such as cancer pain as well as non-cancer pain including various types of neuropathic pain. The departments from which opioids were prescribed were as follow: pain management clinic, internal medicine, family medicine, respiratory medicine, thoracic surgery, orthopedic surgery, otorhinolaryngology, hematology and oncology. From patients whose data were complete for analysis, we excluded female patients because the sample proportion for females was insufficient to analyze gender interactions on the effects of interest . PatientBaseline patient characteristics collected from electronic medical records included age, weight, height, body mass index (BMI), alcohol consumption, smoking, underlying diseases, indication of opioid use, opioid type and maintenance doses, duration of opioid therapy, previous experience of opioid use, concomitant drug use, opioid combination therapy, and OIARs.Only the opioid use from the first visit during our study period (year 2012) was included. Cumulative dose ) was calculated by using conversion factors of 0.15 for codeine and 1.5 for oxycodone. Duration of opioid therapy (defined as continuous use of opioids with a gap of no greater than 30 days) was divided into two groups based on 12 weeks according to Langzeitanwendung von Opioiden bei nicht tumorbedingten Schmerzen (LONTS) Guideline.Previous experience of opioid use was defined as opioid use in the year before their first opioid prescription in 2012. Concomitant drugs during opioid use included tramadol, fentanyl patch, benzodiazepines , amitriptyline, and gamma-aminobutyric acid (GABA) analogues .The primary outcome was to estimate the OIARs in association with baseline and clinical variables. OIAR was defined as the presence of one or more events among GI problems , dizziness, drowsiness, urinary retention, skin rash, respiratory depression, and other symptoms within 4 weeks of opioid prescription. Secondary outcomes were consisted of GI (GI problems) and CNS (dizziness and drowsiness) OIARs.p-values < 0.05 from univariate analysis along with a strong confounder of age (if necessary) were included in multivariable analysis and listwise deletion was used for missing values. Variance inflation factor (VIF) was calculated to evaluate multicollinearity using “Allison’s criteria (VIF>2.5)” [P values less than 0.05 were considered statistically significant. All statistical analyses were carried out using the Statistical Package for Social Sciences (SPSS), version 20.0 for Windows .Chi-squared test or Fisher’s exact test was used to compare categorical variables between patients with OIARs and those without OIARs and unpaired t test were used for continuous variables. Multivariable logistic regression analysis was used to identify independent risk factors for OIARs. The reference group for opioid type in statistical models was defined as codeine, which is less potent than morphine and oxycodone. Factors having IF>2.5)” . Attribun = 81), dyspnea (n = 61), back pain (n = 52), neuropathy (n = 52), post-surgery pain (n = 6), traumatic pain (n = 5), and other pains . OIARs included GI problems (n = 59), dizziness and drowsiness (n = 20), urinary retention (n = 7), skin rash (n = 5), respiratory depression (n = 2), and others .A total of 332 patients were prescribed in the outpatient clinic during the study period. Twelve patients were excluded, including three female subjects (0.9%) and nine subjects (2.7%) with frequent switch of opioids. Accordingly, data from 316 patients who took opioids were used for final analysis. Indications of opioid use were cancer pain (2). Frequencies of alcohol intake and smoking were 95 (32.3%) and 72 (24.2%), respectively. Drugs concurrently administered with opioids included tramadol or fentanyl patch (n = 143), benzodiazepines (n = 92), amitriptyline (n = 87), and GABA analogues (n = 160). Various opioids were used, including oxycodone (n = 111), codeine (n = 94), and morphine (n = 78).As shown in Table Presence of diabetes mellitus (DM), duration of opioid therapy, benzodiazepine use, GABA analogue use, opioid type, opioid combination therapy, and MME were significant factors of OIARs in univariate analysis Tables  and 2. TP value of less than 0.05. Patients with opioid use ≥12 weeks had a 79% increased odds of OIAR compared to those with opioid use < 12 weeks (OR = 1.79). Attributable risk of GABA analogues was 64~78% in constructed Models. Compared to codeine (the least potent) users, patients using morphine and oxycodone had 653 and 473% increased odds of OIAR, respectively. MME ≥ 60 mg/day showed a 317% increased odds  of OIAR compared to MME < 60 mg/day. Opioid combination therapy had a 139% increased odds of OIAR compared to monotherapy.Multivariable analysis showed that duration of opioid use, concomitant use of GABA analogue, opioid type, opioid combination therapy, and MME were significant factors of OIARs after controlling variables with In analysis of GI AR Table , similarIn the case of CNS AR Table , GABA anBoth short-term and long-term use of opioids is commonly associated with multiple adverse effects . Across Opioid-induced bowel dysfunction (OIBD) is a well-known and frequent event, with estimated prevalence of 15 to 90% based on a recent systematic review . Our stuOne of the main findings of our study was that concomitant use of GABA analogue such as pregabalin and gabapentin increased the odds of OIARs by 180~355%. It increased the odds of CNS adverse events by 553~881%. This result suggests that close monitoring for potential risks of OIARs with co-prescribing GABA analogues is warranted. Until recently, gabapentin and pregabalin have been considered to have a low profile of drug interactions. They have been used as adjunctive drugs to opioids for clinical management of neuropathic and chronic pain . NotablyMore than half of our study patients received co-prescription of opioids with GABA analogues. In comparison, a combination of opioids with other CNS depressants such as benzodiazepines and amitriptyline was used in less than one third of our patients. This might be because their additive effects on sedation have been well recognized. The American Geriatrics Society (AGS) has been emphasizing adverse drug interactions between opioids and CNS active drugs including benzodiazepines and tricyclic antidepressants . ReportePotential harms and benefits of dual-opioid therapy are under increasing discussion. In our study, administration of opioid combination increased the odds of both all OIARs and GI AR by 130% compared to monotherapy. There are controversial issues in terms of efficacy and safety of morphine-oxycodone combination for both acute and chronic pain . At the Although less pronounced, the frequency of all OIARs and GI adverse events in our patients was increased with long-term therapy (12 weeks and over). In addition, MME over 60 mg resulted in attributable risk of 76 and 70% in all OIARs and GI adverse events, respectively. Recent studies have also shown that higher opioid dose is associated with the risk of OIARs such as fractures, depression, opioid overdose, and misuse . One stuA couple of reviews have been performed to address whether one opioid is superior to another with respect to tolerability. It has been claimed there are no differences in the incidence of adverse effects between morphine and meperidine . Other aOur study has several limitations. Since our dataset solely came from adverse events detected by chart review, rates of OIARs appeared to be lower compared to those by incident reporting or controlled trials . MoreoveNevertheless, our findings have important clinical implications. Due to pharmacokinetic and pharmacodynamic differences compared to younger patients, elderly patients may have an increased risk of OIARs. Also, older patients exhibit a higher likelihood of polypharmacy and drug-drug interactions with opioids.Our data suggest that concomitant use of GABA analogue, strong opioids, and dual-opioid therapy may increase the risk of OIARs in the elderly. Low dose short-term use showed less risk of OIARs in our study population. Therefore, it is recommended that opioids be prescribed at the lowest effective dose to minimize OIARs, and clinician should carefully monitor patients when starting opioid therapy in older population.


Lawsonia intracellularis is an anaerobic obligate intracellular bacterium infecting the small intestine and infrequently also the large intestine of pigs and other animals including hamsters and horses. The infection is characterized by proliferation, hemorrhage, necrosis, or any combination commonly referred to as “ileitis,” affecting the health and production efficacy of farmed pigs. Despite decades of research on this pathogen, the pathogenesis and virulence factors of this organism are not clearly known. In pigs, prophylaxis against L. intracellularis infection is achieved by either administration of subtherapeutic levels of in-feed antibiotic growth promoters or vaccination. While the former approach is considered to be effective in L. intracellularis control, potential regulations on subtherapeutic antibiotics in many countries in the near future may necessitate alternative approaches. The potential of manipulating the gut microbiome of pigs with feed ingredients or supplements to control L. intracellularis disease burden is promising based on the current understanding of the porcine gut microbiome in general, as well as preliminary insights into the disease ecology of L. intracellularis infection accrued over the last 30 years. Lawsonia intracellularis (L. intracellularis) is prevalent worldwide and the infection manifests in pigs in two clinical presentations  or porcine PE (PPE), is commonly observed in weaned and growing pigs less than 4 months of age. It is associated with decreased weight gain and low mortality due to proliferation and thickening of the ileum and the proximal colon. Acute L. intracellularis infection is known as proliferative hemorrhagic enteritis (PHE), and is characterized by intestinal hemorrhages and sudden death, usually occurring in mature pigs older than 4 months , was characterized as Gram-negative and acid-fast positive using modified Ziehl Neelsen staining  nor in equine or rabbit isolates, it may not be required for pathogenicity in pigs but rather be a feature of adaptation to the porcine host  is expressed in cell culture and in infected ileal tissue, and was identified using degenerate primers , lscO, and lscQ genes, a family of virulence genes found in many bacteria which together encode a putative type 3 secretion system, were found to be expressed in infected pig intestinal epithelial cells (L. intracellularis autotransporter protein A (LatA), was identified based on mass spectrometric analysis of cell culture expressed L. intracellularis proteins that reacted with infected pig sera , aldehydes , oxidizing agents (Virkon® S), biguanindes (Nolvasan® Solution), phenol (Tek-Trol®), iodine (Cetridine), chlorine, potassium peroxymonosulfate, phosphate compounds , and sulfate compounds effectively inactivate arbonate –45. An iarbonate .in vitro culture system for L. intracellularis, screening for antimicrobial sensitivity using the rat enterocyte-based culture system  showed penicillin, erythromycin, difloxacin, virginiamycin, and chlortetracycline had the highest activity to inhibit bacterial multiplication followed by tiamulin and tilmicosin  is dominated by Prevotella, and enterotype 3 (or F) is dominated by Firmicutes, most prominently Ruminococcus  and Allium sativum (garlic) in pig feed produced reduction in L. intracellularis load in the intestine and a decrease in clinical disease, with improved production parameters in a herd with history of L. intracellularis infection  and bacteriocins that modulate the gut microbiota, gut morphology, immune system, and other beneficial effects –96. In additives . Other rdditives , 96. Feet health . Feed teted pigs , 98. Expnfection –100. Exp litters . These p litters . Anothermed pigs . Though nfection . Similarllularis . The maxLactobacillus to manipulate the human gut microbiome was reported in 1921 (Enterococcus faecium was shown to suppress diarrhea and mortality induced by pathogenic Escherichia coli in pigs (L. intracellularis, addition of lactic acid to feed and fermented liquid feed partially mitigated the pathogenesis and shedding of organism in farmed pigs (Escherichia coli Nissle, considered a probiotic organism, enhances barrier function of intestinal epithelia and improves protection against rotavirus in pigs; a mechanism which could work in the context of L. intracellularis infection (L. intracellularis infection (Bacillus spp are known to elicit beneficial innate immune responses in the gut epithelium and also have systemic effects on the immune system (L. intracellularis infection is an avenue left unexplored until now.The benefits of fermented food for human consumption, with or without live microbes, in improving gut health and controlling enteric pathogens has been appreciated since ancient times , 107. A  in 1921 . Enteroc in pigs . In regamed pigs . Probiotmed pigs . Certainmed pigs –115. Escnfection . Some minfection , 117. Otnfection . CompounL. intracellularis was first cultured in vitro and 15 years since the genome was sequenced, but understanding of the pathogenesis of this bacterium is still not conclusive. However, beneficial attenuated oral vaccines and inactivated vaccines are available to pig producers. The host response to L. intracellularis infection is being gradually unraveled, but the mechanism of L. intracellularis pathogenesis and the virulence factors of the bacterium are not yet definitively known. Information obtained from analysis of gene expression patterns of pathogenic and nonpathogenic L. intracellularis and infected host cell gene expression profiles need to be further explored. The proliferative lesions observed with clinical L. intracelluaris infection are not yet replicated in any in vitro infection model. The inability of pure culture of L. intracellularis to establish infection in gnotobiotic pigs remains an unexplained observation and would lend clues to the microbial milieu that favors L. intracellularis to establish infection. While PPE is observed in weaned and growing pigs and PHE is observed in pigs over 4 months of age, the reason behind this variation in pathogenesis with age is not known. Hamsters, which show susceptibility to the pig L. intracellularis and develop pathological lesions similar to that in pigs, could be utilized as a model to further study the disease ecology of L. intracellularis under gnotobiotic conditions. Understanding the dynamics between the gut microbiome and L. intracellularis infection will help in formulating appropriate diet-based control of the disease, which would be synergistic with current vaccines.It has been 25 years since AK drafted the manuscript and both AK and TO reviewed the literature and revised the final manuscript.The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest.


Exposure to high intensity noise produced by MRI is a cause for concern. This study was conducted to determine the temporary and permanent effects of exposure to noise created by performing MRI on the hearing threshold of the subjects using conventional and extended high frequency audiometry. This semiexperimental study was performed on 35 patients referred to Shahid Rahnemoun Hospital for head and neck MRI due to different clinical conditions. The hearing threshold of patients was measured before, immediately after, and 24 hours after performing 1.5 Tesla MRI using conventional and extended high frequency audiometry. SPSS version 18 was used to compare the mean hearing thresholds before and after MRI using paired T test and repeated measures analysis. Comparison of auditory thresholds in conventional and extended high frequencies before and immediately after MRI showed a significant shift at 4 KHz (P = 0.008 and P = 0.08 for right and left ears), 6 KHz (P = 0.03 and P = 0.01 for right and left ears), and 14 KHz (P =0.03 and P = 0.31 for right and left ears). However, there was no significant difference between audiometric thresholds before and 24 hours after MRI. Noise due to 1.5 Tesla MRI can only cause transient threshold shift. High levels of noise can temporary impair hearing thresholds and cause temporary threshold shift (TTS) which is probably reversed after abstinence from noise exposure. Permanent threshold shift (PTS) occurs after a mechanical injury to hair cells due to exposure to noise , 2. AccoMagnetic resonance imaging (MRI) is an ever-increasing imaging modality used to detect many lesions in the body. New MRI devices, due to their detailed imaging and three-dimensional measurements, are widely used for central nervous system imaging. MRI device produces noise during imaging which is positively related to the strength of magnetic field (in Tesla) and may affect patient's hearing –6. StudiHead and neck MRI especially may affect hearing due to the closeness of the device to the ear. Hearing damage after exposure to MRI noise depends on the frequency and intensity of the noise and duration of the exposure and also the distance between ear and MRI device .Some previous studies have found that MRI may affect hearing by oxidative stress and cochlear hair cells injury .The noise produced by MRI devices is dependent on MRI strength, so that the MRI devices with different powers (0.2 to 3 Tesla) produce varying noise levels from 101 to 131 dBA . The freThe studies about the effect of MRI noise on hearing thresholds are few and controversial. Some case reports have found MRI as the cause of TTS , 12 and This study was performed to define the effect of noise produced by 1.5 Tesla MRI on hearing thresholds in different audiometric frequencies (500-16000 Hz) in patients referred for head and neck MRI.This was a before-after study performed in Shahid Rahnemoun Hospital. Participants were selected by consecutive sampling from patients referred to MRI center of Shahid Rahnemoun Hospital to perform head and neck MRI with different indications during January till December 2017. An informed consent was obtained from each participant. Patients older than 50 years were not selected and those with moderate hearing loss (hearing threshold higher than 40 dB at each frequency) and conductive hearing loss were excluded from the study after performing baseline audiometry.MRI device was a Siemens  with magnetic field intensity of 1.5 Tesla. Five steps were performed for each imaging: localizing (10 s), T1 axial (90 s), T2 axial (120 s), T2 trim (180 s), T2 sagittal (90 s), T2 sagittal (90 s), and T2 coronal (90 s) with 1-2-second interval between each protocol. Totally, imaging lasted about 10 minutes for head and 7 minutes for neck.Pure-tone audiometry (PTA) was done using a diagnostic audiometer  by an expert audiologist for each participant at three occasions: (1) 10 minutes before imaging (baseline); (2) during 1 hour after imaging (to detect TTS); and (3) between 24 and 48 hours after imaging (to detect PTS). Hearing threshold for air conduction (AC) and bone conduction (BC) was measured for each frequency in each ear separately. Hearing frequencies which were tested by PTA included 500, 1000, 2000, 3000, 4000, 6000, 8000, 10000, 12000, 14000, and 16000 Hz.Data were analyzed by SPSS ver. 19 using paired T test and repeated measures analysis.Initially 62 patients between 14 and 45 years old were selected. After the first audiometry, 29 patients were excluded due to moderate hearing loss or conductive hearing loss, and at last 33 patients continued the study.Mean (±SD) of age was 31 (±9.7) years (range: 16-45). Threshold shift was observed after MRI and the highest threshold shift one hour after MRI was observed at 4 KHZ and 14 KHz in conventional and extended high frequency audiometry in both ears. Hearing damage due to loud noise can be temporary or permanent. Many individuals experience a TTS after exposure to loud noise which disappears some hours after the termination of exposure; but in some individuals the hearing loss may exist even after several hours of abstinence from exposure to noise. In permanent hearing loss, hair cells of the organ of Corti are damaged due to some mechanisms.MRI is a widely used imaging method in different disciplines of medicine and due to its advantages, its application is being increased. Exposure to high levels of noise is one of the problems which the patients may experience during imaging and this loud noise may cause TTS or PTS. Wagner et al. found that MRI produces a sound pressure level at the patient's ear between 79.5 to 86.5 dBA and sort-term peaks up to 120 dB .In this study, we examined the effect of noise produced by 1.5 Tesla MRI on hearing status of the patients. We assessed both TTS and PTS at conventional and extended high frequencies. The results showed that TTS was observed at 4, 6, 8, and 14 KHz frequencies, but this threshold shift disappeared after 24 hours, so no PTS was observed.The results of the studies on hearing loss after MRI are controversial. Most studies have assessed TTS at conventional frequencies , 12, 13.The results of the present study showed that bilateral hearing loss was observed at 4, 6, and 14 KHz which are the most common frequencies affected by continuous noise in occupational settings –20. HearRadomskij et al. found that 1.5 tesla MRI caused 68% more hearing changes measured by OAEs in patients not wearing ear plugs compared those wearing ear plugs . They diIn the present study, patients had authority to use hearing protection devices and some of them used them. Jin et al. used ABR for assessing the effect of 3 Tesla MRI noise on hearing and found only TTS in the patients who wore hearing protection devices, which was consistent with the results of the present study .This study showed that noise produced by 1.5 Tesla MRI during head and neck MRI probably causes TTS without permanent hearing threshold shift.


This study was conducted to determine the dynamic Islet1 and Brn3 (POU4F) expression pattern in the human fetal retina and human-induced pluripotent stem cell- (hiPSC-) derived retinal organoid. Human fetal eyes from 8 to 27 fetal weeks (Fwks), human adult retina, hiPSC-derived retinal organoid from 7 to 31 differentiation weeks (Dwks), and rhesus adult retina were collected for cyrosectioning. Immunofluorescence analysis showed that Islet1 was expressed in retinal ganglion cells in the fetal retina, human adult retina, and retinal organoids. Unexpectedly, after Fwk 20, Brn3 expression gradually decreased in the fetal retina. In the midstage of development, Islet1 was detected in bipolar and developing horizontal cells. As the photoreceptor developed, the Islet1-positive cone precursors gradually became Islet1-negative/S-opsin-positive cones. This study highlights the distinguishing characteristics of Islet1 dynamic expression in human fetal retina development and proposes more concerns which should be taken regarding Brn3 as a cell-identifying marker in mature primate retina. Islet1, also known as ISL1, is a LIM-homeodomain transcription factor that plays critical roles in differentiation, cell specification, and phenotype maintenance of horizontal cells as well as cholinergic amacrine and ganglion cells in the retina of different species including fish, reptiles, birds, amphibians, chickens, and mammals. Moreover, numerous studies have revealed that ISL1 plays a key role in multiple tissue types, such as the heart , skeletaSince 2012 , studiesAn essential question regarding retinal organoids is whether they mimic human fetal eye development. If so, Islet1 may be useful as an indicator for determining the developmental process of retinal organoids compared to the human fetal retina.To evaluate Islet1 dynamic expression in human fetal retina and hiPSC-derived retinal organoid development, we characterized subsets of Islet1-positive cells based on morphological features and performed coimmunostaining with specific markers of retinal neuron subtypes. We determine the expression profile of Islet1 during human fetal retina development and examined variations in the developmental process in retinal organoids. During the study on Islet1, Brn3 showed distinguished express pattern from a rodent model. Therefore, the Brn3/Pou4f family of transcription factors, well-known markers of RGC, is also investigated in the present study.Human fetal eyes ranged in age from 8 to 27 fetal weeks (Fwk) and were obtained from legal routine therapeutic abortions at the Third Affiliated Hospital, Sun Yat-sen University. Fetal age was determined by eye size and foot length. The human adult retina slices were collected by the pathology department of Zhongshan Ophthalmic Center, from the samples for pathology examination and choosing the peripheral normal retina. The retinoblastoma tissue was collected by Prof. Rong Lu. Rhesus slices were collected from the control eyes of a previous study . All samThe retinal organoids were differentiated, as described previously , from thHuman fetal eyeballs < Fwk 15 were fixed with 4% paraformaldehyde in 0.1 M PBS (pH 7.2–7.3) for 2–4 h according to the size of the eyeballs at room temperature (24-26°C). In elder eyes, the anterior segment  and vitreous were removed to obtain posterior cups for fixation. Residual eyecups were fixed overnight at 4°C. Retinal organoids were collected and fixed in 4% paraformaldehyde for 0.5 h.μm  or 14 μm , which were collected onto SuperFrost plus slides .Next, fetal eye tissues and retinal organoids were rinsed in 0.1 M PBS at room temperature to remove residual fixative and cryoprotected with a sucrose gradient  in phosphate buffer . Subsequently, the samples were embedded in optimal cutting temperature (OCT) compound (OCT: 25% sucrose = 1 : 2). A Leica CM1850 cryostat  was used to produce serial sections at thickness of 10 α antibody , anti-Recoverin antibody , anti-Rhodopsin antibody , anti-Ki67 , anti-S-opsin , and anti-L/M-opsin , which were donated by Jeremy Nathans .OCT compound was removed by a 15 min incubation in 37°C PBS, and cryosections were incubated in permeabilization and blocking solution (PB) containing 0.2% (vol/vol) Triton X-100 and 10% (wt/vol) normal donkey serum in PBS for 1 h at room temperature. Primary antibodies were incubated with the sections at 4°C overnight. The following primary antibodies were diluted in 2% (wt/vol) donkey serum, 0.04% (vol/vol) Triton X-100 in PBS: anti-Islet1 antibody , anti-MCM2 antibody , anti-VSX2 antibody , anti-Brn3 antibody , anti-Brn3a antibody , anti-RPBMS antibody , anti-HuD antibody , anti-AP2μL ProLong® Gold Antifade Reagent with DAPI was applied 10 minutes at room temperature to counterstain the nuclei. Subsequently, all samples were rinsed in PBS and mounted in Vectashield and coverslipped. H&E staining was performed as described previously [Primary antibody staining was followed by three washes with 1x PBS. Subsequently, cryosections were incubated for 1 h at room temperature with secondary antibodies that included the corresponding species-specific Alexa Fluor-488-, Alexa Fluor-555-, and Alexa Fluor-657-conjugated antibodies . Next, 2 eviously , 15.t test Prism Software Version 7 .Images were acquired on an Olympus BX53 microscope  and Zeiss . Confocal images were acquired with a Zeiss LSM 510 confocal microscope. Immunofluorescence intensity was evaluated by ImageJ , and for statistical comparisons, values were subjected to a two-tailed Student's -ΔΔCt method. The expression levels of target genes were normalized to that of GAPDH. Primer sequences are listed in Total RNA was isolated using TRIzol reagent (Sigma-Aldrich) from fresh tissue and RNAprep pure FFPE kit  from formalin-fixed tissue, and RNA quality was evaluated with a NanoDrop1000 spectrophotometer (Thermo Fisher Scientific). The first-strand cDNA was synthesized with a PrimeScript RT Master Kit  according to the manufacturer's instructions. Quantitative PCR was performed using a LightCycler 480 SYBR Green I Master  on a LightCycler 480II system (Roche). Reactions were performed in triplicate, and Ct values were calculated using the 2th fetal week (Fwk 8), the retinal ganglion cell layer (GCL) became organized, leading to a gap between the GCL and the neuroblast layer. Up to fetal week (Fwk) 16, the neuroblast layer became thicker and a thin gap began to form inside of this layer ).is layer ″). In Dwis layer ″). WhethTo determine whether Islet1 colocalizes with early markers of RGC specification, coimmunolabeling with anti-Islet/-Brn3 and anti-Islet/-HuD was performed. In the early weeks, Islet1 was mainly expressed in Brn3-positive retinal ganglion cells. However, at the most basal side, Islet1 expression was higher, while on the outer side, clusters of Brn3-positive but Islet1-negative cells were detected . In the Double staining with HuD, which is also an RGC marker as demonstrated by Romero-Aleman and colleagues , showed Costaining with another common RGC marker, RPBMS, it was shown that Islet1, HuD, and Brn3 could be coexpressed in RGC during Fwk 16 to Fwk 22 Figures , as wellOn the mRNA level, Brn3a expression was significantly low in adult human and rhesus retina as referred to hiPSC. Brn3b and c were not detectable in qPCR in adult human retina. Interestingly, during retinal organoid development, Brn3b expression was the main expression subtype among Brn3a, b, and c, while in human fetal retina, Brn3a was the major . ConsideIn Fwk 16, Brn3a was stained in GCL , and to Interestingly, in the early stage, HuD protein was expressed mainly in the cytoplasm Figures . From Fwα-positive cells were also located in the INL. According to a study by Bassett et al., AP2α is exclusively expressed in postmitotic amacrine cells during retinal development and in mature amacrine cells in the adult retina [AP2t retina . From FwProx1, a developing horizontal cell marker, colocalized with Islet1  is expressed in retinal progenitor cells. As shown in Figures ar cells . In the At Fwk 15, a mononucleus layer of Islet1-positive cells was observed. When double labeled with Recoverin, some cells were colocalized Figures . AdditioDouble staining with Rhodopsin and Islet1 revealed that the Islet1-negative photoreceptors were rods Figures . The IslWe evaluated the dynamic expression of Islet1 in the human fetal retina and compared the hiPSC-derived retinal organoid with human fetal retina development. The results revealed that Islet1 temporal and spatial expression was generally similar to that in various vertebral animal models. However, we also found the Islet1 was specifically expressed in cone precursors of the human fetal retina. Further, the retinal organoid showed the same patterns of Islet1 expression as the fetal retina, indicating its potential as a development model and drug-screening model. However, in later stages of development, the retinal organoid showed a developmental delay compared to the human fetal retina in the specification of photoreceptors and retinal lamination.Xenopus laevis. In the present study, we examined the retina of human embryos. The Islet1 spatiotemporal expression in human fetal showed a very similar pattern to in mammals, from ganglion to amacrine and bipolar cells. Further, the “retina-in-dish” showed a similar pattern of expression.A recent study by Bejarano-Escobar et al.  confirmeConsistent with a study by Prasov and Glaser , human fWe found that Brn3 expression gradually decreased during fetal development. The RGC in adult human retina could not be stained by Brn3 antibody, as in the adult primate retina. Then, we investigated the mRNA level of Brn3a, b, and c during the development of fetal retina and adult retina. In adult retina, Brn3b or c was not detectable, while Brn3a was 135-fold higher, consistent with Whitmore's study by RNA-seq that POU4F1 (Brn3a) was enriched in the macular part of retina . After nMoreover, HuD protein  is an RGC marker . In contα-positive amacrine cells in retinal organoids gained their fate during a similar period as in the human fetal retina, they failed to migrate and organize in later development. In contrast, the development of bipolar cell delayed, but the cells organized into a layer in INL.Correspondingly, double labeling of Islet1 and amacrine- and bipolar-specific markers in the human fetal retina and retinal organoid showed the same pattern as in the mouse retina, chicken retina, etc. , 42–44. Few studies have focused on Islet1 and photoreceptor specification. According to Wang et al., Islet1-/Recoverin-positive cells were cone precursors , which wIn conclusion, this is the first study to describe the Islet1 and Brn3 expression pattern throughout development in the human fetal retina. The expression of Islet1 in subsets of retinal neurons was mainly consistent across species from fish to human, supporting that Islet1 plays a critical role in human retinal cell specification, differentiation, and maintenance. Additionally, we determined the full picture of development and cell specification in hiPSC-derived retinal organoid comparing to in the fetal retina.in situ hybridization. Finally, compared to the fetal retina, the hiPSC-derived retinal organoid has three main limitations: RGC was not preserved in later development, RGC transcript profile differed from those in fetal retina, and the lamination and organization were not distinct. Therefore, the differentiating protocol must be optimized, possibly by using additional transcription factors.There were some limitations to this study. First, although Brn3 and Brn3a did not stain RGCs in the adult primate retina, whether Brn3b and c proteins were all covered by present antibody was not clear. Due to the shortage of samples, quantification of protein was not able to be performed in the present study. Thus, it was unclear how many Brn3 transcript factors would translate into protein. Therefore, the role of Brn3 and Islet1 in RGC differentiation in primate requires further analysis. Second, a previous study demonstrated that the Islet coexpressed with cone markers was Islet2, rather than Islet1 . Thus, a


Coffee is the one of the most common beverages worldwide and has received considerable attention for its beneficial health effects. However, the association of coffee with hearing and tinnitus has not been well studied. The aim of this study was to investigate the association of coffee with hearing and tinnitus based on a national population-based survey. We evaluated hearing and tinnitus data from the 2009–2012 Korean National Health and Nutrition Examination Survey and their relationship with a coffee consumption survey. All patients underwent a medical interview, physical examination, hearing test, tinnitus questionnaire and nutrition examination. Multivariable logistic regression models were used to examine the associations between coffee and hearing loss or tinnitus. We evaluated 13,448 participants (≥19 years) participants. The frequency of coffee consumption had a statistically significant inverse correlation with bilateral hearing loss in the 40–64 years age group. Daily coffee consumers had 50–70% less hearing loss than rare coffee consumers, which tended to be a dose-dependent relationship. In addition, the frequency of coffee consumption had an inverse correlation with tinnitus in the 19–64 years age group but its association was related with hearing. Brewed coffee had more of an association than instant or canned coffee in the 40–64 years age group. These results suggest a protective effect of coffee on hearing loss and tinnitus. Coffee is the most commonly consumed beverage, apart from water, in the world . Coffee Coffee contains over 1000 bioactive compounds ,15 with Hearing loss is a major public health problem ,19. One-Tinnitus is not a single disease entity. Actually, it is a symptom that decreases quality of life and is related with hearing loss and aging. The prevalence of tinnitus is 12–30% worldwide ,25. In aThe aim of this study was to investigate the association of coffee with hearing and tinnitus in adult and elderly participants based on a national population-based survey. We compared the consumption frequency and type of coffee and the prevalence of hearing loss and tinnitus.This study used data from the Korean National Health and Nutrition Examination Survey (KNHANES). This survey collects information, such as health and nutritional status, from a representative sample of the general Korean population to assess the health-related behavior, health condition and nutritional state of Koreans.The subjects were asked about their hearing, symptoms of tinnitus, health behavior and nutrition by questionnaire. The participants were asked about the annoyance of tinnitus measured by the following answers: “No,” “slightly annoying” and “very annoying and difficult to sleep.” Information about the subjects included sleep time, stress, education level (less than middle school or beyond high school), education level of the parents, income , current smoking status and alcohol drinking status . Health status  was also checked. Duration of occupational exposure to noise and earphone and headphone use time were measured.Physical examinations were conducted by a physician to assess any problems with the tympanic membrane or other ear, nose and throat problems, including perforation or retraction of the tympanic membrane, otitis media with effusion and cholesteatoma. Pure tone audiometry was performed at 0.5, 1, 2, 3, 4 and 6 kHz in a soundproof room. The severity of hearing loss was based on a lower threshold of unilateral hearing loss and a higher threshold for bilateral hearing loss. The pure tone average was the average of the hearing levels at 0.5, 1, 2 and 3 kHz or 0.5, 1, 2 and 4 kHz, whereas the high frequency hearing level was the average of the hearing levels at 3, 4 and 6 kHz. Blood samples were collected and analyzed in a single laboratory .In total, 36,067 individuals participated in the 2009–2012 KNHANES. Individuals with ear disease  were not included here. Of them, 27,492 participants were age ≥19 years. Among 27,492 participants aged ≥19, 9294 participants were excluded because they did not complete all three component surveys  (n = 4480) or examined from January 1 to July 20 in 2009  or aged ≥ 65 in 2012 . Of the remaining 18,198 subjects, additional 4750 were excluded because they did not receive hearing threshold testing nor respond to tinnitus-related questions (n = 975) or did not respond for coffee consumption frequency (n = 629) or have missing values for covariates considered in this study (n = 3146). Finally, 13,448 subjects  were included in the analysis for the present study . This stCoffee consumption frequency was assessed using the food-frequency questionnaire (FFQ). Participants were asked to indicate how frequently they consumed coffee over the previous year based on ten categories  in 2009–2011 and on nine categories  in 2012, in which the first two categories in the previous FFQ version were combined into “never or seldom” and four times per week was grouped into two or three times per week.Coffee consumption frequency was categorized into rarely, monthly, weekly and daily using the FFQ data as follows: rarely, less than once per month; monthly, one to three times per month; weekly, one to six times per week; and daily, once or more per day.The information on the types and amount of all coffee that participants consumed over the past 24 h was collected by trained dietitians 1 week after the health interview. The type of coffee was grouped into brewed, instant, or canned coffee using the 24-hour dietary recall method.The subjects’ characteristics according to coffee consumption frequency are presented as median (interquartile range) or number (proportion) and compared using the Fisher exact test (binary covariates), the chi-square test (more than three categories), or the Wilcoxon rank-sum test (continuous covariates). Multivariable logistic regression models were used to examine the associations between coffee consumption and hearing loss or tinnitus. The analyses were adjusted for the following potential confounders: age, sex, education, parents’ education, perceived stress, exposure to indoor secondhand smoke, current smoking, heavy drinking, drinking-related problems, menopause, history of hypertension, diabetes mellitus, anemia, kidney failure, thyroid disorder, tympanic membrane perforation, cholesteatoma and otitis media with effusion. The multivariable models for tinnitus or annoyance related to tinnitus included hearing loss as well as the potential covariates described above. To examine the association of the type of coffee consumed with hearing loss and tinnitus, multivariable logistic regression analyses were performed for the adjusted associations between coffee type consumed and hearing loss or tinnitus. All statistical analyses were performed using SAS software .The prevalence rates of unilateral and bilateral hearing loss in the study population were 1.19% and 0.17% for subjects in the 19–39 years age group, 5.01% and 2.9% for subjects in the 40–64 years age group and 14.24% and 20.97% for subjects in the ≥65 years age group. The prevalence rates of tinnitus and tinnitus-related annoyance were 18.07% and 3.86% for subjects in the 19–39 years age group, 19.92% and 6.24% for subjects in the 40–64 years age group and 27.98% and 12.82% for subjects in the ≥65 years age group . The parp = 0.0021), whereas monthly or weekly consumers did not show a significant difference relative to rare consumers. In the 40–64 years age group, odds ratio of mild and moderate hearing loss in daily coffee consumers and mild hearing loss in weekly coffee consumers were significantly lower than those of rare coffee consumers (onsumers . In addip = 0.0186) and 40–64 years (unadjusted odds ratio (OR), 0.81; 95% CI, 0.67–0.99; p = 0.0357) age groups. However, in the multivariable models adjusted for potential confounders, the relationships between daily coffee consumers and rare consumers were not significant in the 19–39 year  and the 40–64 years age groups . An inverse association was observed between tinnitus-related annoyance and coffee consumption in weekly coffee consumers aged 19–39 years  and daily coffee consumers aged ≥ 65 years . However, the associations in weekly coffee consumers aged 19–39  and daily coffee consumers aged ≥ 65 years  were not significant in the multivariable analysis.p = 0.0028). And the odds ratio of tinnitus for brewed coffee in 19–39 years age group is significantly lower than 1 . Contrary, the odds ratio of tinnitus for canned coffee in 40–64 years age group is significantly higher than 1 .We investigated associations between types of coffee and hearing loss, tinnitus and tinnitus-related annoyance using multivariable analysis. This study demonstrated the inverse correlation of the frequency of coffee consumption with hearing loss in middle aged Koreans. The prevalence of bilateral hearing loss in daily coffee consumers was significantly lower in the 40–64 years age group compared to the other age groups. However, no significant correlation was observed between unilateral hearing loss and coffee consumption in any other age group. Tinnitus and tinnitus-related annoyance were not related with coffee consumption. Instant coffee consumers aged 40–64 years had less hearing loss and less tinnitus-related annoyance than those in the ≥65 years age group and for any type of coffee.No previous large-scale study has demonstrated the effects of coffee on hearing loss and tinnitus. Many people believe that coffee has a harmful effect on hearing. Actually, there is a report that caffeine in coffee has detrimental effects on recovery from acoustic overstimulation events ,32. CaffOur univariable analyses determined that the prevalence of tinnitus in daily coffee consumers in the 19–39 and 40–64 years age groups was lower than that in rare coffee consumers of the same age groups. The prevalence of tinnitus in weekly coffee consumers age ≥65 years was lower than that in rare coffee consumers in the same age group. However, this tendency disappeared in the multivariable analysis, suggesting that coffee consumption itself does not have a direct correlation with tinnitus. Some covariates, such as bilateral hearing loss, stress and sleep, can indirectly affect tinnitus.The relationship between coffee and tinnitus is controversial. There is an opinion that caffeine in coffee stimulates ascending auditory pathways or reduces the suppressive effect on the central nervous system, which evokes tinnitus . Other sThe relationship between the frequency of coffee consumption and the occurrence of tinnitus in the 19–39 and ≥65 years age groups appeared to be quite different from that of the 40–64 years age group. The abatement of tinnitus in the 40–64 years age group can be explained by a decrease in bilateral hearing loss. However, no significant decreases in bilateral hearing loss were observed in the 19–39 years and ≥65 years age groups. One of the covariates, such as perceived stress, can reduce tinnitus. In fact, an inverse correlation has been reported between perceived stress and tinnitus ,47,48. CTypes of coffee have association with hearing loss and tinnitus. Our results suggest that brewed coffee can have preventive effects on bilateral hearing loss and tinnitus but canned coffee can have inducing effect on tinnitus for some age groups. Difference in preparation method, heat treatment (freeze-drying or high temperature sterilization) and expiration date seem to have affected on bioactive constituents in each type of coffee ,50. HoweWe have some limitations in this study. First, this is an observational study, so it is difficult to generalize the result of this study to the causal relationship from coffee consumption to hearing loss and tinnitus. To confirm causality, well-controlled experimental design will be needed. Second, we could not analyze unilateral and bilateral tinnitus separately, because KNHANES data did not discern tinnitus side. However, bilateral tinnitus is different from unilateral one in the point of heritability and prognosis . TherefoAccording to the results of KNHANES analyses, coffee consumers had a low prevalence of bilateral hearing loss. However, the path of lower bilateral hearing loss and tinnitus varied according to age group. The incidence of bilateral hearing loss was low in coffee consumers aged 40–64 years, which influenced the low prevalence of tinnitus. However, other covariates, such as the low perceived stress of coffee consumers, seemed to be the main cause for the low prevalence of tinnitus in the 19–39 and ≥65 years age groups. In addition, brewed coffee consumers had lower rate of bilateral hearing loss and tinnitus.


Stable cesium has been used to understand the long-term behavior of radionuclides in plants, trees and mushrooms. We are interested in using 133Cs to predict the future transfer factor (TF) of radiocesium from contaminated logs to shiitake mushrooms in Eastern Japan. However, the current methodology to obtain a representative wood sample for 133Cs analysis involves mechanically breaking and milling the entire log (excluding bark) to a powder prior to analysis. In the current study, we investigated if sawdust obtained from cutting a log along its length at eight points is as robust but a faster alternative to provide a representative wood sample to determine the TF of 133Cs between logs and shiitake.Stable cesium  and fruiting bodies (collected separately from each disc), and the wood-to-shiitake TF was calculated.Oak logs with ready-to-harvest shiitake fruiting bodies were cut into nine 10-cm discs and each disc was separated into bark, sapwood and heartwood and the concentration of 133Cs did not differ (P = 0.223) compared to either the sapwood-to-shiitake TF or heartwood-to-shiitake TF, but bark did have a higher concentration of 133Cs (P < 0.05) compared to sapwood and heartwood. Stable cesium concentration in sawdust and fruiting bodies collected along the length of the logs did not differ (P > 0.05).We found that the sawdust-to-shiitake TF of 133Cs. To satisfy the goals of different studies and professionals, we have described two sampling methodologies (Methods I and II) in this paper. In Method I, a composite of eight sawdust samples collected from a log can be used to provide a representative whole-log sample , whereas Method II allows for the simultaneous sampling of two sets of sawdust samples—one set representing the whole log and the other representing wood only. Both methodologies can greatly reduce the time required for sample collection and preparation.Sawdust can be used as an alternative to determine the log-to-shiitake TF of  Lentinula edodes) production in Japan (134Cs and 137Cs) in general foods, including mushrooms cultivated on logs, to ≤ 100 Bq/kg fresh weight (FW) (137Cs concentration in xylem wood (120 Bq/kg) will occur in 2039, compared with 6–47 Bq/kg found in contaminated oak sapwood in 2012  . The traght (FW) . A provight (FW) . Becauseght (FW) , it is pght (FW) . In the ght (FW)  and be tght (FW)  giving rght (FW) . It has  in 2012 . AlthougThe consumption of food contaminated with radionuclides is a significant route of radionuclide intake by humans . Althoug133Cs analysis because the current in-house methodology is both time and labor intensive  would result in an underestimation of the sawdust-to-shiitake TF. Third, we determined the 133Cs concentration in sawdust and fruiting bodies along the length of the logs. The distribution of 133Cs will have implications for the number of sawdust and fruiting body samples needed torepresent the entire log and mushroom crop, respectively. Based on the findings of this study, we propose two different sampling methodologies using sawdust to determine the log-to-shiitake TF of 133Cs.Our goal in this study was threefold. First, we tested if the sawdust-to-shiitake TF of t growth , little f a tree  such as ca. 10-cm intervals along its length (n = 9 discs/log). The sawdust produced from cutting each disc (n = 8 sawdust samples/log) and fruiting bodies present on each disc (n = 9 fruiting body samples/log) were collected and analyzed for 133Cs concentration. For five of the 10 logs, each 10-cm disc was separated into bark (n = 9 samples/log), sapwood (n = 9 samples/log) and heartwood (n = 9 samples/log) and also analyzed for 133Cs concentration. An overview of the sampling method is shown in Ten oak logs and their ready-to-eat shiitake fruiting bodies were sampled. Each log was cut at Quercus serrata “konara” is the most common oak species grown in Eastern Japan and commonly used for shiitake cultivation in Japan. Logs with shiitake mushrooms were supplied by a grower located in Ibaraki Prefecture, Japan. This grower purchases up to 200,000 logs per year from oak suppliers throughout Japan. The grower reported that the logs used in the current study were sourced from trees felled in either December 2016 or January 2017 and allowed to dry for 1 month before inoculation. Logs were inoculated with multiple 2-cm plug spawns in either January or February 2017. Fruiting occurred in a humidity- and temperature-controlled environment with logs stored in a horizontal orientation. To minimize the influence of possible confounding factors affecting 133Cs distribution in logs and fruiting bodies, all logs used in this study were (1) from the same species of oak (Q. serrata), (2) sourced from the same region , (3) inoculated with the same strain of shiitake , (4) managed similarly from inoculation to fruiting, and (5) producing their first crop of fruiting bodies which were ready to collect and eat on the day of sampling. Selected logs needed to have at least one fruiting body present on each 10-cm interval along its length.Logs and fruiting bodies were collected in March 2018. For each log, 10-cm intervals along its length were demarcated and labeled. Fruiting bodies growing on each 10-cm disc were collected, the bottom 1 cm of the stipe was removed with a knife to eliminate any attached pieces of bark and then the fruiting bodies were placed into factory-new paper bags. When sampling was complete, logs and fruiting bodies were transported to the laboratory.ca. 2–3 mm) before being placed loosely on a polypropylene mesh, dried overnight at 60 °C and then at 105 °C for 1 day and reweighed to determine the dry weight (DW). The use of a mesh avoided the fruiting bodies adhering to the inside of the paper bags while drying and the initial drying at 60 °C ensured the sliced fruiting bodies were not too hard for milling. The dried fruiting bodies were milled using an Iwatani mill to a powder and placed into air-tight factory-new plastic bags which in turn were placed into a larger air-tight plastic bag containing silica gel sachets.Fruiting bodies from each 10-cm disc were weight and thinly sliced (ca. 1 day). To determine DW of wood and bark, each 10-cm disc was first dried at 60 °C for 2–3 days and then at 105 °C until the weight became constant (ca. 3 days), and then each disc was separated into the bark, sapwood and heartwood parts using a hammer and chisel. We distinguished the heartwood from the sapwood by the darker color of the heartwood. Bark, sapwood and heartwood samples were crushed using Horai V-360 mill (Japan) and all parts (including sawdust) were milled to a powder using an Iwatani mill. Samples were placed into air-tight factory-new plastic bags which were then placed into a larger air-tight plastic bag containing silica gel sachets.The weight, length and diameter of each log was recorded. Discs were cut from each log using a circular saw  and the sawdust was collected using a dust collector connected to a vacuum. The wet and DW of each sawdust, wood and bark samples were recorded and their DW determined. Each sawdust sample was placed into a separate pre-labelled factory-new plastic container, dried for 1 day at 60 °C and at 105 °C until the DW became constant  rather than where it was likely to be low , multiple wood, bark and fruiting body samples were collected from each log and no analytical replicates were used. To confirm the soundness of this decision, five randomly selected samples representing the materials in this study were digested in duplicate and the 133Cs concentration measured in each. The deviation in 133Cs concentration between these duplicate digested samples was very low compared to the variation observed within logs  were digested in 10 ml of HNO3 (60–61% (mass/mass); Wako, Japan) using the Multiwave 3000 digester ; samples were digested at 600 W for 40 min at ≥120 °C (30 bars of pressure). The digestates were diluted 1-in-5 with Milli-Q water  and filtered  prior to 133Cs analysis. The 133Cs content was measured by inductively coupled plasma-mass spectrometry  with cesium standard solution  and indium (two µg/l) as the internal standard . To minimize variation between ICP-MS runs, wood, bark, sawdust and fruiting bodies of individual logs were analyzed in the same run. At the time of ICP-MS analysis, a subsample (0.5 g) of each sample was oven dried at 105 °C for 48 h for residual moisture determination. For quality control purposes, the National Institute of Standards and Technology standard reference material 1575a (Pine needles) was included in each analysis. Average precision for 133Cs was 8.5%. Stable cesium values were determined on a DW basis unless stated otherwise.Fruiting bodies, log parts and sawdust samples  and even-numbered discs  were compared with the paired sample t-test. To demonstrate that the whole-log TF and wood-only TF can be determined simultaneously, the TF based on sawdust collected from odd-numbered cuts  was compared with sawdust from even-numbered cuts  (t-test.To demonstrate that collecting fruiting bodies from four of the nine discs (per log) would be representative of the shiitake crop on a log, 4, 6, 8)  using th133Cs concentrations in log parts, sawdust and shiitake fruiting bodies and their level of significance were determined. All statistical analyses were carried out using the SPSS version 25 for Mac . Values were deemed significantly different when P < 0.05.Correlation coefficients of The mean and SD in weight, length and diameter of the 10 logs was 5 ± 1.0 kg, 94 ± 0.9 cm and 9 ± 1.0 cm, respectively. The logs and fruiting bodies had a DW of 601 ± 41.4 and 109 ± 6.8 g/kg, respectively. There were on average 36 fruiting bodies per log.P > 0.05) than both the sapwood-to-shiitake TF and heartwood-to-shiitake TF. The mean concentration of 133Cs in the sapwood and heartwood was lower  than in the bark (133Cs concentrations in sawdust (P > 0.05) between cuts and discs, respectively. Stable cesium concentration in fruiting bodies collected from odd- (600 µg/kg DW) and even-numbered discs (581 µg/kg DW) did not differ (P > 0.05) (P > 0.05)   and the  > 0.05) . Stable 133Cs. The current in-house methodology to ensure thatwood and shiitake fruiting body samples for inorganic elemental analysis are representative involves cutting logs into nine 10 cm-discs, discarding the bark and mechanically breaking the wood into smaller pieces , and then compositing the wood from three discs to provide a total of three subsamples per log for 133Cs analysis  and 4.2 (on a FW basis). To the best of our knowledge, this is the first report of the TF of 133Cs between logs and shiitake, although the TF of radiocesium was reported to be 0.43 between semi-dried logs (12% moisture content) and fresh fruiting bodies  in this study, the numerically lower TF value based on sawdust reflects the higher concentration of 133Cs in the bark. For shiitake cultivation, logs from trees aged ca. 15–25 years are used, and therefore it was important to confirm if 133Cs distribution between heartwood, sapwood and bark showed a similar trend as found in older oak trees.In the current study, we found that the TF based on 133Cs in sawdust and fruiting bodies along the length of the logs did not differ significantly and there were no unusually high concentrations in any one sawdust sample or in fruiting bodies from any one disc in individual logs  and then mix these samples to produce one representative sample for the whole log. Comparing 133Cs concentration in fruiting bodies collected from odd- and even-numbered discs, we found no significant difference between these two sets of discs  across different treatments.Bark comprises 10–20% of woody vascular plants  and this133Cs as a proxy element to understand the long-term behavior of radiocesium, plant and mushroom physiologist may prefer to use a TF of 133Cs based on wood only. Whereas in Japan, the government requires researchers in the food industry to report the TF of 133Cs based on the whole log. These preferences are likely based on either tradition or belief of where shiitake sources its 133Cs rather than from empirical evidence.When using The three factors discussed above highlight the need for a more comprehensive and flexible sampling methodology. Below, we describe a second sampling methodology (Method II) to help satisfy the goals of different studies and professionals.133Cs concentration between heartwood, sapwood and bark  and found no significant difference . Althougand bark  would inand bark  and 4C. ca. 11 and ca. 63 min to sample a log of similar size using Method I (The current study did not compare the time and labor efficiencies obtained from collecting sawdust samples (Methods I and II) over wood samples  because it was obvious from the outset that major savings in both parameters would be found. However, from our experience sampling logs, we estimate that approximately 146 min is needed by one technician to sample a single 20 cm-diameter shiitake-inoculated log  using the in-house sampling method . In contMethod I  and 3E aMethod I  and 4C, 133Cs from logs to shiitake. The use of sawdust will greatly reduce the time for sample collection and preparation, and this will facilitate sampling a larger number of logs in Eastern Japan to predict the future TF of radiocesium from contaminated logs to shiitake. To further refine the two proposed sampling methodologies discussed, it would be important to determine the proportion of 133Cs in shiitake fruiting bodies originating from bark and wood and its availability in each part.Collecting multiple sawdust and fruiting body samples per log is a robust and efficient method to provide a representative sample to determine the TF of 10.7717/peerj.7825/supp-1Supplemental Information 1Click here for additional data file.


The retina is a thin membrane derived from the neuroectoderm, it is the physical morphological substrate in which the transformation of light energy into electrical impulses, that later will be led to the cerebral cortex, is performed. Due to its prosencephalic embryological origin, the retina is normally considered a specially differentiated part of the brain. It is a very complex tissue, formed by multiple cell layers and by several types of neuronal cells , microglia (macrophages), macroglia , and vascular cells (endothelium and pericytes). Under physiological conditions, the retina is characterized by a high oxygen consumption rate, intense exposition to pro-oxidizing agents  and a high content of polyunsaturated fatty acids . Therefore, retina is especially susceptible to oxidative stress ,2,3.Oxidative stress is defined as the imbalance between the generation and elimination of reactive oxygen species (ROS) and it results from either excessive ROS or an impaired antioxidant system. To cope with ROS increase, the retina has evolved different antioxidants defenses such as vitamin E, ascorbate, catalase, glutathione (GSH), glutathione-peroxidase, and glutathione-transferases . HoweverOxidative stress has been related with a great number of retinal pathologies including diabetic retinopathy, age-related macular degeneration (AMD), glaucoma, and retinal dystrophies ,6,7,8,9.All possible retinal malfunctions and diseases will severely affect the quality of life of patients because the retina allows the phenomenon of vision that is essential for the development of everyday life, and damage to the retina, as a part of the visual system, can lead to blindness.The hypothetical role of oxidative stress in the development of retinal diseases has given rise to research on the use of antioxidants as preventive and therapeutic agents in retinal therapy. Although the use of antioxidants seems promising to improve results in eye disorders, more research, mainly related to bioavailability, distribution, and interactions between antioxidant molecules, is necessary to standardize the indications for its use, composition, and dose. This Special Issue consists of a set of articles which highlight different aspects of antioxidants and retinal tissue in health and disease. These papers illustrate the role of substances or molecules such as saffron, quercetin, ethylmethylhydroxypyridine malate, coenzyme Q10, abscisic acid, progesterone, lipoic acid, and progesterone, as neuroprotective agents in retinal degeneration situations.Di Marco et al.  suggest Different animal models can be used to study retinitis pigmentosa (RP), a disease with no cure. Among the most used models are the light-induced retinal degeneration model and the rd1 and rd10. Koyama et al.  study thNitric oxide (NO) is a gas molecule with diverse physiological and cellular functions in the retina, however, products generated from NO  and peroxynitrite) have great oxidative damaging effects. Cantó et al.  review tIn conclusion, the studies regarding the use of antioxidants in retinal diseases are complex but support antioxidant supplements as therapeutic aids.


Cordyceps sinensis are still unclear. To explore the mechanisms, proteins potentially related to the fruiting body formation, proteins from fruiting bodies, and mycelia of Cordyceps species were assessed by using two-dimensional fluorescence difference gel electrophoresis, and the differential expression proteins were identified by matrix-assisted laser desorption/ionisation tandem time of flight mass spectrometry. The results showed that 198 differential expression proteins (252 protein spots) were identified during the fruiting body formation of Cordyceps species, and 24 of them involved in fruiting body development in both C. sinensis and other microorganisms. Especially, enolase and malate dehydrogenase were first found to play an important role in fruiting body development in macro-fungus. The results implied that cAMP signal pathway involved in fruiting body development of C. sinensis, meanwhile glycometabolism, protein metabolism, energy metabolism, and cell reconstruction were more active during fruiting body development. It has become evident that fruiting body formation of C. sinensis is a highly complex differentiation process and requires precise integration of a number of fundamental biological processes. Although the fruiting body formation mechanisms for all these activities remain to be further elucidated, the possible mechanism provides insights into the culture of C. sinensis.The fruiting body formation mechanisms of  Cordyceps is a large genus of entomogenous fungi with more than 400 species found world-wide, and the most famous and valuable species is Cordyceps sinensis (Berk.) Sacc. , immature (early stage) fruiting bodies and mycelium of Paecilomyces militaris (Kob.) Brown & Smith ex Liang, anamorph of C. militaris (L.: Fr.) Link  Fr., anamorph of C. memorabilis (Ces.) Sacc.  was gift from Zhangjiagang City Zanglian Biotechnology Co., Ltd., Jiangsu Province, China; and strain Isaria farinosa (anamorph of C. memorabilis) was isolated from infected caterpillar provided by Qinghai Academy of Animal and Veterinary Science, China, which was identified by the Institute of Microbiology of Chinese Academy of Sciences, China. Golden rabbit Thai fragrant rice purchased from San Miu Supermarket Limited in Macao, China; foxtail millet  obtained from Yilan County Seed Company, Heilongjiang Province of China; silkworm larvae and silkworm pupa powder bought from Sericulture and Farm Produce Processing Research Institute, Guangzhou, China; mould liquid medium purchased from Guangdong Huankai Microbial Sci. & Tech. Co., Ltd., China.Natural fresh H. sinensis or 22°C for P. militaris and I. farinose until plentiful mycelia balls presented. The mycelia were harvested by centrifugation, washed twice with sterile PBS buffer, and stored at 4ºC after lyophillisation. The fruiting body of C. militaris, C. memorabilis was cultured in improved rice medium , 50 g silkworm pupa powder, 16.6 g mould liquid medium and 1 l distilled water) sterilised at 121ºC for 30 min. Each cultivation bottle was inoculated with 8 ml of liquid seed and incubated in the dark at 22ºC for C. militaris and C. memorabilis with humidity levels of 70%, respectively. When the mycelia completely colonised the jar, the jar was exposed to fluorescent lamp (about 200 lx). After 3 to 5 days, the temperature was set a cycle of 22ºC for 12 h, and 12ºC for 12 h under a 12 h light/dark cycle condition to promote primordia formation. Once a large number of primordia were produced, the temperature was kept at 22ºC, and the relative humidity was kept at around 85% under a 12 h light/dark cycle condition for the formation of the fruiting body. The early and late stage fruiting bodies of C. militaris were cultured in fifth instar larvae of silkworm. In brief, the 5th instar silkworm larva was surface sterilised with medical povidone-iodine swabs and then 0.3 ml of mycelial homogenate was injected under axenic conditions. The inoculated larvae were fed with fresh mulberry leaves at 22°C in the dark with 70% relative humidity. After the larva grew into stiff silkworm, the cultivation conditions were the same as the fruiting body of C. militaris grown on solid medium. When the fruiting body grew to approximate 2 cm (early stage) or produced spores (late stage), they were harvested and stored at −80°C.he fungi were cultured as the method described in our previous report  were ground to a fine powder in liquid nitrogen using a mortar and pestle, added lysis buffer  CHAPS, 1% (w/v) DTT, 0.5% (v/v) IPG buffer pH 3–10, and 1 mM PMSF, from GE Healthacare) based on the modified Handbook 80–6429-60AC , and continued to grind to homogenate. The homogenate was transferred to a 1.5 ml Eppendorf tube and frozen in liquid nitrogen for 3 min, and then it was thawed in 37°C water for 3 min. For fully extracting the intracellular proteins, this step was repeated three times. After centrifugation at 28,113 × g for 5 min at 4°C. Added co-precipitant 4 times the size of the pellet after removing the supernatant, next centrifuged at 28,113 × g for 5 min. Pipetted enough Milli-Q water to disperse the pellet, and added 1 ml of pre-chilled wash buffer and 5 μl wash additive at −20°C for at least 30 min, vortexed for 20–30 s once every 10 min. The mixture was further centrifuged at 28,113 × g for 5 min at 4°C, and the supernatant was discarded and the pellet was allowed to dry briefly. The pellet was solubilised in lysis buffer without DTT and IPG buffer. Lastly, the protein solution was centrifuged at 28,113 × g for 20 min at 4ºC, and the supernatant was collected and stored at −80ºC. Prior to quantification, pH of protein samples was adjusted to 8.5 by using 1 M NaOH, as monitored by the pH Test Strip . Finally, protein concentrations were determined with Bio-Rad Protein Assay Dye Reagent Concentrate (Bio-Rad) using BSA (2 mg ml−1) as the standard.Proteins were purified with a 2-D Clean-Up Kit  according to the manufacturer’s instructions, revision 80–6486-60/Rev. CO/11–02. Briefly, proteins solution (200 μl) was mixed well with 600 μl of precipitant and incubated for 15 min on ice, then 600 μl co-precipitant was added and centrifuged at 28,113 × All steps were operated in dark room. The proteome samples were labelled for DIGE analysis using Cy2, Cy3 and Cy5 CyDye™ DIGE Fluor minimal dye , respectively, according to the manufacturer manual . Cy2 was used to label an internal standard which was pooled equal amounts of each of all samples. Each 50 μg protein sample was labelled at a ratio of 400 pmol of dye on ice for 30 min, and the labelling reaction was terminated by adding 1 μl of 10 mM lysine and left on ice for 15 min. The three labelled samples were mixed into a single tube, and then both of extra 300 μg paired protein samples in a gel were added to the same tube, thus total of 750 μg protein samples were mixed in the tube and later could be used as preparative gel for spots picking. Equal volumes of 2× sample rehydration buffer , and 0.004% bromphenol blue) was added to the protein samples. Rehydration buffer  was added to reach volumes to 450 μl for rehydration.The mixture was transferred to IPGbox , and ReadyStrip IPG Strips  was put on the mixture with the gel side down. The gel was covered with DryStrip Cover Fluid , and rehydrated for 18 h at 20°C. After rehydration, the IPG strip was transferred to Ettan IPGphor Manifold of Ettan IPGphor 3 Isoelectric Focusing Unit . The first dimension isoelectric focusing (IEF) separation of 2DE was performed at 20°C with following sequential steps: 50 V rapid for 4 h; 150 V gradient for 2 h; 250 V gradient for 2 h; 500 V gradient for 2 h; 1,000 V gradient for 3.5 h; 5,000 V rapid for 1.5 h; 8,000 V rapid for 2 h; 10,000 V rapid for 70,000 Vh. After IEF, the strips were equilibrated in 50 mM Tris-HCl (pH 8.8), 6 M urea, 30% glycerol, 2% SDS, and 0.01% bromophenol blue with the addition of 2% DTT for 15 min in the dark. Subsequently, the strips were equilibrated with the same buffer with 2.5% (w/v) iodoacetamide instead of DTT for 15 min in the dark. Prior to preparation of SDS-polyacylamide gels, the longer low fluorescent glass plate was painted with PlusOne™ Repel-Silane ES  to assure gel release; while the shorter glass plate was painted with 4 ml PlusOne™ Bind-Silane  in 1 ml of acidic ethanol (0.5% acetic acid in 95% ethanol) to covalently attach the polyacrylamide gel to glass surface. SDS-PAGE was performed as second dimensional separation in 12.5% acrylamide gels in an Ettan™ DALT Six-Large Vertical System . The electrophoresis was performed at 15 mA per gel for 40 min, then 30 mA per gel until the bromophenol blue line reached the bottom of the gel at 10°C in the dark.Cy2-, Cy3- and Cy5-labelled samples were acquired in an Ettan DIGE Imager  according to the manufacturer’s instructions. The images were checked for intensity during the acquisition process using ImageQuant™ TL software , and analysed by using DeCyder™ 2-D Differential Analysis Software v7.0 . After analysis, the gels were stained in 0.1% Coomassie Brilliant Blue-R250 solution in 50% ethanol and 10% acetic acid for 2.5 h, and destained in 25% ethanol and 8% acetic acid for 1.5 h. Then, they were washed with Milli-Q water and scanned on a UMAX PowerLook 2100XL scanner (UMAX Technologies). Protein spots at least 2-fold differences in expression level were automatically and accurately excised into 96-cell plates using Ettan Spot Picker . All parameters were set according to the manufacturer’s protocol.TM 384-well Insert, Applied Biosystems), and 0.4 μl of matrix solution (saturated solution of α-cyano-4-hydroxycinammic acid in 50% acetonitrile and 0.1% TFA) were added to the peptide and allowed to air-dry at room temperature. Tryptic peptides of 0.5 μl were analysed using a 4800 plus MALDI TOF/TOF Analyser (Applied Biosystems) with positive ion reflection mode, and standards  were conducted to calibrate the spectrum to a mass tolerance within 0.1 Da. The parameters for database searching were peptide tolerance of 80–150 ppm; MS/MS tolerance of 0.2–0.3 Da; one missed cleavage; variable modifications of carbamidomethyl (Cys), oxidised (Met). GPS Explorer™ software v3.6 (Applied Biosystems) was used to search files in the National Centre for Biotechnology non-redundant (NCBInr) all species database, fungus database and insect database. All the identified proteins have MASCOT report total protein score C.I.% or total ion C.I.% greater than 95% and identification probability score at p < 0.05.Each excised protein slice was washed with 100 μl Milli-Q water  on ice, and destained with 80 μl of 50 mM ammonium bicarbonate (Sigma) /acetonitrile  (1:1) for 30 min at 37°C. The gel piece was incubated in 70 ul of acetonitrile until it was white and sticky. After removing solution, the gel piece was rehydrated in 2 μl of trypsin solution (2 μg Promega’s Sequencing Grade Modified Trypsin mixed with 140 μl of 25 mM ammonium bicarbonate containing 10% acetonitrile) on ice for 20 min. Excess trypsin was removed, the gel piece was covered with 20 μl of 25 mM ammonium bicarbonate containing 10% acetonitrile, and trypsinised at 37°C for 16 h. After digestion, the peptides were transferred into a new PCR tube, and the pellet was extracted with 30 μl of 0.1% trifluoroacetic acid  in 67% acetonitrile at 37°C for 30 min, subsequently was ultrasonicated for 20 min at room temperature. This step was repeated again. Total extracts and the first digested peptides were mixed, vacuum-dried, and dissolved in 2 μl of 0.1% TFA in 30% acetonitrile. After in-gel digestion, 0.4 μl of the supernatant was spotted onto a MALDI plate  from natural C. sinensis were used, and 2100 spots were detected in both the fruiting body and sclerotium (C. sinensis (Jin C. sinensis (Threshold mode: 2.0-fold). Among the detected proteins, only 62 proteins in 70 spots (36.8%) out of 190 picked spots  (Though nsis Jin  and 630 nsis Kao . Among t.0-fold) , and 64 .0-fold)  were suc.0-fold) . There w.0-fold) .C. militaris is a major species of Cordyceps widely used in the market. The fruiting body of Paecilomyces militaris, anamorph of C. militaris, is easily formed in cultured media. It is great help to know the proteins expression during fruiting body formation based on the investigation of proteins in mycelia, early and late stages of the fruiting body of C. militaris. As a results, 2175 protein spots were detected in both early and late stage fruiting bodies of C. militaris, 295 (13.6%) and 234 (10.8%) out of the detected spots were up-regulated and down-regulated in the late stage fruiting body, respectively (Threshold mode: 2.0-fold)  and 449 (20.2%) of detected spots were up-regulated and down-regulated, respectively, in the fruiting body of C. militaris (Threshold mode: 2.0-fold) (C. militaris (.0-fold) . Among 9.0-fold) , 40 prot.0-fold) . On the .0-fold) . Finally.0-fold)  were sucilitaris .C. memorabilis is one of the species of Cordyceps genus. The fungus, Isaria farinose, anamorph of C. memorabilis, could form the fruiting body under laboratory conditions. Therefore, comparison of proteins in the fruiting body and mycelia of I. farinose is also helpful to well understand the molecular mechanism of formation of natural C. sinensis. By DIGE analysis, 2275 protein spots were detected in both the fruiting body and mycelia of I. farinosa. Among the detected spots, 554 (23.9%) and 430 (18.9%) spots were, respectively, up-regulated and down-regulated in the fruiting body (Threshold mode: 2.0-fold) (.0-fold) , and 23 .0-fold)  were suc.0-fold) .Cordyceps . It was worth to note that enolase/putative enolase up-regulated, while ATP synthase down-regulated coincidentally in all fruiting bodies of Cordyceps.Totally, 115 differential expression proteins in 134 protein spots were found in both fruiting bodies and mycelia of three species of C. sinensis, acetaldehyde dehydrogenase, beta-tubulin, elongation factor 2, enolase, malate dehydrogenase, heat shock 70 kDa protein and hypothetical protein FG09893.1 were simultaneously up-regulated in both fruiting bodies of C. sinensis versus sclerotia and fruiting bodies of C. sinensis versus mycelia of H. sinensis. These results suggested that the seven proteins played important roles during the fruiting body formation of natural C. sinensis. Besides, the enolase or putative enolase was up-regulated, while ATP synthase was down-regulated coincidentally in fruiting bodies of C. memorabilis and C. militaris than that in corresponding mycelia of I. farinosa and P. militaris, which showed that the enolase and ATP synthase were the most important proteins for fruiting body formation of Cordyceps. The beta-tubulin was also up-regulated in the fruiting bodies of C. militaris as well as in natural C. sinensis. Although malate dehydrogenase and acetaldehyde dehydrogenase were up-regulated in fruiting bodies of natural C. sinensis, they were down-regulated in the fruiting bodies of C. memorabilis.For natural C. sinensis versus sclerotia, and fruiting bodies of C. sinensis versus mycelia of H. sinensis. The reasons may arise from different samples  and a small probability of picking the same spot in different gels under blind screening .Apart from the shared proteins, most of the differential expression proteins were non-shared proteins in the fruiting bodies of Fruiting body formation of filamentous fungi is one of the most complex developmental processes. It not only requires the aggregation of hyphae to form three-dimensional structures, and leads to the differentiation of a number of fruiting bodies-specific cell types not present in the vegetative mycelium  and mitogen-activated protein kinase (MAPK) pathway, regulate gene expression that finally leads to fruiting body formation. Indeed, MAPK genes are required for fruiting in C. sinensis  highly express during fruiting body formation and development. On the other hand, Hsps are also immunodominant antigens and major targets of host immune response during different types of infection , were shown higher expression in the fruiting body of sinensis . It was ilitaris . These rStigmatella aurantiaca  and aldehyde dehydrogenase are putative indole receptor proteins involved in multicellular development which are essential for fruiting body formation in ilitaris  suggesteC. sinensis was near 13-fold higher than that in mycelia  in the fruiting body, which is a developmental regulation enzyme involving in trehalose, cellulose and glycogen synthesis in fungus lerotium , which mC. sinensis rather than in mycelium  in the fruiting body of natural C. sinensis than that (14.8%) in fermented mycelium  during fruiting body formation , which was over-expressed in mature ascoma of fungus Tuber borchii, might be involved in its fruiting body formation  of Giardia and that its allosteric anabolic activation may shift the equilibrium of this pathway towards UDP-Gal-NAc synthesis (Bulik et al. N-acetylglucosamine pyrophosphorylase was increased in the fruiting body of C. sinensis (UDP-sinensis , which wsinensis .A. bisporus (De Groot et al. A. bisporus (De Groot et al. Septin, as a cell division control protein, is involved in septa formation during cell division, and the highest expression of septin is found in the transitional zone between cap and stipe of mature mushroom Antrodia cinnamomea (Chu et al. Mago nashi proteins, highly conserved among eukaryotes, not only participate in oogenesis, embryogenesis and germ-line sex determination during animal development, but also play important roles in pollen tube growth, root development and spermatogenesis during plant development (Chen and Chu C. sinensis, besides higher septin was in the mature fruiting body of C. militaris (In this study, septin, mago nashi protein and cell division control protein 3 distinctly increased in the fruiting body of ilitaris . These iCordyceps, and 24 proteins have been proven their roles in fruiting body development in other fungi (C. sinensis. Especially, enolase and malate dehydrogenase were first proposed in fruiting body development of mushroom. Besides, the cAMP signal pathway as well as glycometabolism, protein metabolism, energy metabolism, cell division and cell reconstruction are presumed to be related to fruiting body development of C. sinensis (C. sinensis was also hypothesised (C. sinensis is a highly complex differentiation process and requires precise integration of a number of fundamental biological processes. Although the fruiting body formation mechanisms for all these activities remain to be further elucidated, the study presented here provides a framework for understanding them.In summary, this study identified 198 differential expression proteins that may relate to fruiting body development of er fungi . Among tsinensis . A map othesised . It has


Based on our results we make suggestion toward use examined smoothness measures. In total the log dimensionless jerk proved to be the most appropriate in ADL, as long as trial durations are controlled.Smoothness is a main characteristic of goal-directed human movements. The suitability of approaches quantifying movement smoothness is dependent on the analyzed signal's structure. Recently, activities of daily living (ADL) received strong interest in research on aging and neurorehabilitation. Such tasks have complex signal structures and kinematic parameters need to be adapted. In the present study we examined four different approaches to quantify movement smoothness in ADL. We tested the appropriateness of these approaches, namely the number of velocity peaks per meter (NoP), the spectral arc length (SAL), the speed metric (SM) and the log dimensionless jerk (LDJ), by comparing movement signals from eight healthy elderly (67.1a ± 7.1a) with eight healthy young (26.9a ± 2.1a) participants performing an activity of daily living (making a cup of tea). All approaches were able to identify group differences in smoothness , three revealed high to very high sensitivity , three showed low within-group variance , two showed strong correlations between the first and the second half of the task execution (intra-trial R Despite the great importance of analyzing ecologically valid activities in clinical research and diagnostics, the quantification of activities of daily living (ADL) was typically limited to subjective scorings of videos , 2 or tiFor different types of movement tasks, trajectories and corresponding velocity profiles differ in the signals' structures . AssessiIn this study we assessed the validity of different smoothness parameters by comparing the hand movements of young and elderly participants in an ADL. We investigated to what degree the used parameters were sensitive, variable, and independent of general movement characteristics like velocities, trial durations, path lengths, or the activity level. Further, we examined if the parameters are quantifying a general ability to produce smooth movements. ADL offer a way to examine behavior in the context of disease and aging in an ecological valid way and should therefore be considered in research and clinical assessment , 21, 32.We compared the movements of the dominant hand of 8 healthy young adults (26.9a ± 2.1a) with the movements of the dominant hand of 8 healthy elderly participants (67.1a ± 7.1a). The ADL task was to unimanually prepare a cup of tea with milk and sugar . All parThe experimental set-up was similar to the one in Gulde et al. . ParticiThe positional data of the dorsum of the hand was obtained by a Qualisys motion capturing system  incorporating 5 Oqus cameras sampling with a frequency of 120 Hz. There were no gaps in the recordings. All post-processing was computed with MatLab . After differentiation the data were smoothed using a 0.1s local regression filter (“loess”) . This shThe used smoothness parameters were the spectral arc length , the ne, the spec] being the frequency band and V(ω) the Fourier magnitude spectrum /mean(z-scores) (with = 1 being perfectly stable and < 0 being perfectly unstable). The z-standardization was performed in order to be able to compare the sensitivity between the different parameters. Multiple linear regression models were applied to check for independence from the following kinematic parameters: trial duration .The smoothness parameters were compared between groups using ohen's d . Sensitiduration , path leduration , mean peduration , and relduration . The criduration  as r > 02s ranging from 0.22 to 0.91. Note, that only the speed metric and the log dimensionless jerk reached significance and delivered power estimates of at least 0.80. The models of multiple linear regression showed significant models for number of velocity peaks per meter, spectral arc length, and log dimensionless jerk. The models for speed metric were all non-significant , although the sample size was small with 16 participants. The small sample size could have led to missing a possible dependence of the speed metric on kinematic parameters. Each of the four parameters was strongly connected with at least one other smoothness parameter  further revealed that three of the parameters were significantly correlated between the two halves with the strength of the correlations being strong. Note, that in one case the statistical power was lower than 0.80 . High correlations between the two halves support a generalization beyond this specific ADL. By splitting, two different tasks were artificially created and in case of high intra-trial correlations, the metric shows the capability to estimate the participant's general and not task restricted movement smoothness. Lastly, the models of multiple linear regression revealed an impact of kinematic parameters on three of the parameters. All of the models were strong (Table p = 0.07) and the model of multiple linear regression revealed a strong dependence on the traveled path length and mean peak velocity. The impact of mean peak velocity on smoothness can be explained by the fact that smooth movements promote faster movements and the change in the signal-to-noise ratio  can deliver appropriate estimates of movement smoothness in complex motor tasks like ADL. Future research should examine the different sub-types of those measures and different ADL tasks to see if the behavior of the parameter class and the ADL (tea making) are generalizable.The search for a universal smoothness parameter for ADL should have a high priority in neurorehabilitational research in order to assess motor capacity and supervise the rehabilitation process of patients suffering from neurological diseases like stroke, Parkinson's disease, or cerebral palsy. So far, comparisons of movement smoothness with control groups or in patient groups with high (kinematic) variabiliy and during the supervision of the rehabilitation process with changes in trial durations, mean peak velocities, or path lengths, are limited. A promising approach could be using wavelet transformation in order to estimate the complexity of the signal .There are clear limitations in this study. Although examining an ADL, ecological validity was limited by the unimanual execution of the task. However, previous research has shown that the transfer from bimanual to unimanual performance has no interaction with age , and thePG and JH designed the study. PG performed the lab testing and the kinematic and statistical analyses. All authors contributed to the coordination of the study and the final draft of the manuscript.The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest.