Jaundice is common among neonates and if untreated can lead to kernicterus. Diagnosing neonatal jaundice (NJ) using Kramer’s method  is considered user-friendly in resource-limited areas. However, there are conflicting findings on reliability of the Kramer’s method in the diagnosis of NJ, particularly of black descent. Therefore, study aimed to determine the accuracy of Kramer’s method in comparison to the total serum bilirubin (TSB) test in the diagnosis of NJ among neonates of black descent in Tanzania.p < 0.05 was considered to be statistically significant.A cross-sectional study was conducted between June and July 2020 at Muhimbili National Hospital (MNH) in Dar es Salaam Tanzania. A total of 315 neonates were recruited consecutively. In each neonate, jaundice was assessed using Kramer’s method and TSB test. NJ A total of 315 neonates were recruited i. A 2 X 2 table was created for the determination of sensitivity, specificity, positive predictive value (PPV), negative predictive value (NPV), positive and negative likelihood ratios (+LR/−LR), and diagnostic accuracy (effectiveness) of Kramer’s method. Cohen kappa (κ) was used to analyze the agreement between Kramer’s method and TSB. Association between independent variables and presence of jaundice were assessed using the chi-square test and the The prevalence of NJ was 49.8% by Kramer’s method and 63.5% by TSB. The Sensitivity, Specificity, PPV, and NPV of Kramer’s method were 70.5, 86.1, 89.8, and 62.6%, respectively. The +LR and –LR were 5.07 and 0.34, respectively. The diagnostic accuracy of Kramer’s method was 76.1%. There was a moderate agreement between Kramer’s method and TSB results . No significant relationship was observed between the independent variables and the presence of NJ.Kramer has a good positive predictive value. However, due to low sensitivity and NPV one cannot say that overall predictive ability is good. Also, clinical assessment by Kramer’s method should not be used for screening of NJ. Further studies are needed to investigate the utility of other non-invasive techniques in detecting NJ among neonates of black descent. Neonatal jaundice (NJ) affects one in two neonates worldwide  and the Early detection/diagnosis and appropriate management of NJ are very crucial in order to prevent its complications. Visual assessment (Kramer’s rule), transcutaneous bilirubinometry (TcB), and TSB are the approaches used for the detection of NJ .Kramer’s method is user friendly because it is non-invasive, does not require any machine and so can be used effectively in resource limited settings. The technique was introduced by Kramer, and showed a positive correlation between progressions of skin discoloration and serum bilirubin levels , 6 due tControversial findings on reliability of this technique for detecting NJ have been reported worldwide. Studies conducted in Pakistan 2010) and Indonesia 2017) reported that Kramer’s method is reliable in detection of NJ and darker skin color is not a matter of concern  reported. However0 and IndDespite those conflicting results, Kramer’s method is the backbone in diagnosis of NJ in most of the low-level health facilities in low and middle income countries like Tanzania. Therefore, this study aimed to determine the sensitivity, specificity and accuracy of Kramer’s method in detecting NJ in comparison with TSB test in Tanzania setting.A hospital-based cross-sectional study aimed at assessing the reliability of the Kramer’s method in detecting NJ was conducted between June and July 2020 at Muhimbili National Hospital (MNH), Tanzania. MNH is a national hospital located in Dar es Salaam center, receiving patients from different parts of the city and regions of Tanzania. The neonatal unit at MNH is one the biggest in the country, serving up to 500 neonates per month. The unit has adequate numbers of skilled pediatricians, medical doctors and nurses. In addition, the MNH has a well-equipped central laboratory with capacity of performing numerous tests, including total serum bilirubin.Black descent neonates aged less than 28 days who were admitted at MNH and whose mothers/guardians provided written consent to participate in the study were enrolled. Neonates with rashes and those who were severely sick  were excluded because assessment of NJ using Kramer’s method cause physical discomfort to the babies.n = Z2x Specificity x (1-specificity) / e2 x ). Specificity of Kramer’s method of 89% from a study conducted in Indonesia [A total of 315 neonates were recruited into this study. The formula for ‘diagnostic sample size calculation’ was used to calculate the sample size  (i.e. n Data were collected using case report form (CRF) comprising of 3 sections; demographic information , Kramer scores and TSB results. The CRF was developed following literature review and experts’ consultation. Each neonate was subjected to visual assessment using Kramer’s method and TSB test. Neonatal jaundice was defined by yellowish discoloration of the skin by visual assessment or TSB level of ≥ 85 μmol/l by TSB .Two medical doctors and 1 pediatrician with experience in neonatal assessment performed the visual assessment on routine ward round. For each neonate, visual assessment using Kramer’s method was performed by the 2 doctors separately and discrepancies were resolved by the pediatrician. Visual assessment of the 5 dermal zones were scored as no jaundice =0, jaundiced at level of head and neck = 1, jaundiced at upper trunk (above umbilicus) =2, jaundiced at lower trunk and thighs (below umbilicus) =3, jaundiced at arms and lower legs =4, jaundiced palms and soles = 5 . The disAbout 2–2.5 mls of blood sample was withdrawn from each neonate’s femoral vein using 2.5 cc syringe into a green topped heparinized vacutainer tube. The collected sample was transported to the laboratory for processing within 2 h using a cooler box containing ice packs. In the laboratory, the sample was centrifuged using a centrifuging machine to obtain serum. The obtained serum was analyzed for bilirubin concentration using the spectrophotometric chemistry analyzer . Bilirubin determination was based on the run of bilirubin with diazotized sulfanilic acid. Azobilirubin concentration was measured at an absorbance of 500-600 nm which is proportionate to bilirubin concentration.2 test) and p < 0.05 was considered to be statistically significant.Data was entered and analyzed by SPSS version 23 software. Frequencies and percentages were used to summarize categorical variables and continuous variables were summarized by using median (interquartile ranges (IQR)). For Kramer’s method, those who scored 0 were grouped as no jaundice while 1–5 were considered to have jaundice. TSB test of ≥85 μmol/l were considered to have jaundice and below that as no jaundice. Sensitivity, specificity, predictive values and diagnostic effectiveness (accuracy) were obtained by contingency Tables (2 × 2 table for diagnostic test) . Diagnos580 neonates were admitted at the MNH neonatal unit during the study period. 247 were not eligible because written consent was not obtained as mothers were left at health facilities where they gave birth due to obstetric complications/management and 333 were enrolled. Out of 333, 18 were excluded because blood samples were not taken  and TSB Of 315 respondents with complete data, 182 (57.8%) were males with the median (IQR) age of 4 [p = 0.46), gender (p = 0.77), delivery mode (p = 0.13), birthweight (p = 0.19) and gestational age (0.78) were not significantly associated with the occurrence of NJ.The prevalence of NJ was 63.5% by TSB and 49.8% by Kramer’s method. The median (IQR) TSB for neonates who were clinically not jaundiced was 65(40–106) μmol/l and 178 (121–238) μmol/l for neonates who were clinically jaundiced. Following chi-square test, all variables including age (P < 0.01) Table Kramer’s method had a Sensitivity-70.5%, Specificity-86.1%, PPV-89.8%, and NPV-62.6% in detecting NJ. It had 5.07 LR of detecting jaundice and 0.34 LR of not detecting jaundice among neonates with a diagnostic accuracy of 0.761. Kramer’s method had moderate agreement with TSB test , moderate sensitivity (70.5%), and good specificity (86.1%). Besides, Kramer’s method was found to have good ability of predicting presence of NJ (89.8%).Our findings are consistent with the study conducted in Indonesia, in which sensitivity and specificity of Kramer’s method reported to be 76.92 and 89.47% respectively. This study was conducted among neonates of black descent and the diagnostic accuracy found to be moderate which is in line with findings from various studies that reported the diagnostic accuracy of Kramer’s method to be poor for neonates of black ethnicity compared to other groups . Hence, Our study also demonstrated slightly increases in sensitivity of Kramer’s method when detecting jaundice among term (75.3%) and normal birthweight (80%) neonates and increase in specificity among neonates with low birthweight (89. 4%). These findings are comparable to what was reported in Denmark and Switzerland where sex, birthweight and gestational age were demonstrated to be the determinants of the diagnostic accuracy of the visual assessment technique , 16. TheTcB, a non-invasive technique, can be used to detect NJ at an early stage. In comparison to TSB, this technique is able to detect bilirubin concentration easily, does not cause heel-puncture pain or irritation to the child, and is simple to calibrate. TcB, however, is unable to detect accurate bilirubin concentrations after 10 mg/dl, necessitating TSB confirmation. Furthermore, since the TcB machine costs thousands of dollars, it is not readily available in resource-limited settings like Tanzania. Similarly, since hemoglobin, melanin, skin types, and age all affect readings, most non-invasive methods cannot be used to diagnose severe disease –19.Lastly this study showed that the occurrence of NJ was independent of age, gender, delivery mode, birthweight and gestational age. These findings are comparable to a study done by Aprillia et al. 2017) which reported that gender, skin color and gestational age were not the determinants for the occurance of NJ. However, our findings are inconsistent with a review conducted in USA (2011) which reported that physiological jaundice was expected to peak between 3 and 5 days of life 17 which . The obsThe fact that this study was conducted at the tertiary referral hospital implies that the prevalence of NJ might have been overestimated. Also, Kramer method could be affected by intra- observer variability which was tacked by reconciling individual observations to the level of one-unit difference in scores, and computing Cohen kappa statistics for reliability during data analysis.Kramer has a good positive predictive value. However, due to low sensitivity and NPV one cannot say that overall predictive ability is good. Also clinical assessment by Kramer’s method should not be used for screening of NNJ. Further studies are needed to investigate the utility of other non-invasive techniques in detecting NJ among neonates of black descent.


Beak trimming of turkeys is an animal welfare issue. It can result in acute pain, potential chronic pain, and a change in feeding, drinking, and pecking ability and general behavior. It is still permitted by law in Germany when the intervention is necessary to protect turkeys from feather pecking and cannibalism. In the present study, an alternative method using grinding wheels (blunting disks) that were fitted in the feed pans when the turkeys were six weeks old was tested. The disks were expected to blunt the beak tips during feeding and reduce the severity of pecking injuries. Six hundred male turkeys of three breeds  were housed separately in 24 groups. The birds in 12 groups were beak trimmed, those in the other 12 were not, but received the blunting disk. The results showed a noticeable beak abrasion in birds provided with blunting disk. Injuries and plumage conditions were equivalent between the treatments. Summarized, the blunting method may be an alternative to infrared beak treatment, but its effectiveness should be confirmed under commercial conditions. The blunting method could potentially result in improved animal welfare of turkeys by minimizing acute pain, chronic pain, and injurious pecking.Feather pecking and cannibalism are behavioral disorders that cause animal-welfare-relevant and economic problems. To mitigate these problems, the beaks of conventionally reared turkeys are usually already trimmed in the hatcheries. To find an alternative to beak trimming, we conducted this study with male turkeys of three breeds: B.U.T. 6, B.U.T. Premium and, Auburn (200 turkeys per breed). Half of the birds had infrared-trimmed beaks; the other half had intact beaks. For each treatment combination , 25 turkeys were housed in one section. A screed grinding wheel was installed in each feed pan of the non-beak-trimmed turkeys as of week six to facilitate natural beak abrasion until slaughter. Eight randomly selected turkeys per section were regularly examined to record injuries, plumage condition, and beak dimensions. In addition, 96 beaks from randomly slaughtered birds were examined macroscopically and histologically. The results concerning injuries and plumage condition showed in most cases no differences between the beak-trimmed turkeys and the ones provided with the blunting disks. The histological examinations revealed alterations in only the beak-trimmed birds. We can conclude that the blunting method smoothens the beak during feeding and thus may be a possible alternative to beak trimming. Beak trimming for the prevention of feather pecking and cannibalism has been a long-time topic of discussion, not only in Germany. In July 2015, the Central Association of the German Poultry Industry, Berlin  and the German Poultry Producers Association, Berlin (Verband Deutscher Putenerzeuger e. V.) came to terms with the Federal Ministry of Food and Agriculture, Bonn (Bundesministerium für Ernährung und Landwirtschaft) in a voluntary agreement on the abandonment of beak trimming , “procedThe German poultry industry has been obliged to allow infrared beak trimming only on the first DOL in the hatchery . In addiThe objective of the present study was to further examine blunting as an alternative method to beak trimming, and thus as a way to improve the welfare of turkeys. Blunting is a process that facilitates natural beak abrasion, which means to blunt the sharp tip of the beak to reduce injurious pecking. In nature, turkeys generally forage the ground for food. They are persevering foragers and frequently peck at rough surfaces such as sand, soil or rocks , and thiThis study was performed according to the legal regulations of the German Animal Welfare Act, 2006 (German designation: Tierschutzgesetz—TierSchG) , the GerThe open-barn research unit of the Bavarian State Research Center for Agriculture, Department of Poultry and Education in Kitzingen , Germany, has a total length of 37 m and a total width of 10 m. The poultry house has a concrete floor, concrete side walls and sandwich panels on the roof. In total, 821 one-day-old turkey chicks of the breeding company Moorgut Kartzfehn von Kameke GmbH & Co. KG, Bösel, Germany, were housed on 26 July 2018 . The chicks were 209 B.U.T. 6 male turkeys, 207 B.U.T. Premium male turkeys, 207 Auburn male turkeys and 198 Auburn female turkeys. Both B.U.T. 6 and B.U.T. Premium male turkeys have white plumage. In Germany, B.U.T. 6 male turkeys in conventional rearing are usually fattened as heavy-weight carving turkeys and reach a slaughter weight of about 21.33 kg at an age of 20 weeks . The breAt the hatchery, all chicks had been vaccinated against turkey rhinotracheitis. Half of the chicks of each breed had been beak-trimmed by infrared beam . The settings of the PSP device for all groups were as follows: power 43, guard plate 24/20 as the smallest for turkeys; mirrors and other aids were not used. According to the manufacturer, the device calculates the radiation duration individually depending on the calibration. Female turkeys of the Auburn breed had to be taken on and were raised in the same barn  were composed: V1 = beak-trimmed B.U.T. 6, V2 = non-beak-trimmed B.U.T. 6, V3 = beak-trimmed B.U.T. Premium, V4 = non-beak-trimmed B.U.T. Premium, V5 = beak-trimmed Auburn male turkeys, V6 = non-beak-trimmed Auburn male turkeys, V7 = non-beak-trimmed Auburn female turkeys. 2. So, there were four replicates of three breeds of birds subjected to two beak treatments. Incoming airflow was automated via adjustable shutters, exhaust airflow via three ceiling fans, one energy-saving fan and two continuously adjustable fans for so-called vacuum ventilation. Rearing took place in the right wing (Sections 1 to 12). Each section was supplied with a corner barrier and a gas-fired infrared heater  during the first 14 days, and with two drinking troughs , two feed pans  and additional feed on chick paper and egg carton during the entire rearing phase. Each section was littered down with 26 kg heat-treated, low-dust wood shavings; litter depth was 4 cm . Feeding during rearing took place in two phases . During the first 12 days, a step-down light program with a stepwise reduction in the light phase and intensity was applied. From the first to the seventh day, the light phase was reduced by one hour per day (from 24 h to 17 h per day), and afterwards, the light intensity was reduced (from between 80 and 100 lx to 20 lx) by means of a barn climate computer . The birds were vaccinated on the 15th DOL and in the 6th, 12Biological Performance: At housing and after the first rearing phase (14th DOL), the body weight of all birds in each section was recorded. After Phase 2 (35th DOL), the birds were weighed again and randomly distributed to the 24 sections for fattening . The light intensity was recorded with a luxmeter ; it was measured at animal head level at five locations in each section , always in six directions . The temperature in degrees Celsius and the relative humidity in percent were recorded and saved hourly by four sensors of a data logger  in the barn and one sensor outside of the barn ; the datTM, Roxell®®, Hans Gaab, Wieseth, Germany), and a 30-grit screed grinding wheel  was installed as a blunting disk (Before the beginning of Phase 3 (62nd DOL), the feed pans for rearing were exchanged with feed pans for fattening . The remaining male turkeys and the female Auburn turkeys did not take part in the study and were fattened separately. The previously occupied sections (1 to 12) were completely cleaned and freshly littered down with 26 kg soft-wood shavings before rehousing to create the same initial conditions in both barn wings. Once a week, 13 kg heat-treated, low-dust soft-wood shavings were added in each section. Furthermore, at the beginning of Phase 3, the elevated tiers, measuring 9600 cm2 , were installed in each section, and lucerne briquets  were provided as enrichment material. The drinking troughs were cleaned manually three times per week. The fattening phase consisted of four additional phases, during each of which phase-specific feed , Phase 4 (90th DOL), and Phase 5 (118th DOL), the birds were weighed in groups, the feed weight was adjusted in each section, and eight birds per section were randomly chosen and examined regarding the animal welfare indicators. We evaluated the plumage on the back, wings  and tailPost-mortem Measurements: Both after Phase 5 and after Phase 6, two birds per section were randomly selected and slaughtered at the poultry slaughterhouse of the Bavarian State Research Center for Agriculture  in Kitzingen, Germany. The heads of the birds were vacuum preserved per variant and stored for 24 h at 2 °C for later histological examination at the Bavarian Health and Food Safety Authority  in Erlangen, Germany. The remaining birds were slaughtered at the turkey slaughterhouse “Süddeutsche Truthahn AG Handelsgesellschaft” in Ampfing, Germany. n = 1 thin section per beak tip). The main focus of the histological examination was to assess whether the normal physiological structures of the beak tip were preserved. The examined aspects included the morphology of the keratin sheath (rhamphotheca), the epidermis and the dermis with special emphasis on the nerve fibers and the presence of Herbst- and Grandry corpuscles and the praemaxillary bone. Histological Examination: First, the intact turkey heads were photographed laterally , and theA sample size analysis was carried out during the planning of the experimental setup. All animal welfare indicators were measured on ordinal rating scales. Therefore, these variables were analyzed separately using cumulative logistic regression models for ordinal response variables. The experimental predictors age, breed and blunting as well as the interaction of the latter two were included as ordinary fixed effects. Of the climatic parameters, light intensity and gaseous ammonia content were also included as fixed effects, whereas temperature and humidity could not be included because of their high correlation with age. Section-specific effects were modeled as unstructured random effects for the intercept. The resulting hierarchical cumulative logistic regression models were estimated in a fully Bayesian approach using the Hamilton Monte Carlo approach implemented in the probabilistic programming language Stan  via the p-value below 0.05 was defined as significant.The continuous variables  were analyzed with an SAS software program packet  according to a linear variance model  with the fixed effects breed, blunting, and the interaction breed × blunting. Before the experiment, a p < 0.001) just before slaughter (p = 0.009) than that of B.U.T. 6 turkeys after Phase 6. There were no differences in body weight (p = 0.420) after Phase 6 between beak-trimmed and non-beak-trimmed birds per breed. Compared with the other two breeds, Auburn showed less body weight  was necessary during the whole experimental period. Four birds  had to be taken out because of pecking injuries/cannibalism. Leg weakness with subsequent kick injuries caused by other birds was the main cause of death, especially at the end of fattening. None of the fecal sample screenings after each fattening phase revealed endogenous parasites.The mortality rate was 0.7% during rearing and 4.2% during fattening  perished and 68% (17 birds) were culled during fattening). The following causes of death were differentiated: pecking injuries/cannibalism, heart failure, leg weakness/kick injuries, and other causes. Other causes included, for example, three birds that had died from unknown causes and thus were pathologically examined at the LGL in Erlangen, Germany. The pathological assessment revealed that two birds had a systemic infection with Effects of the experimental and environmental predictors on animal welfare indicators are presented in Regarding the examined welfare indicators, we could show that beak-trimmed Auburn turkeys had significantly fewer body injuries than beak-trimmed turkeys of the other two breeds . In comparison, among the non-beak-trimmed turkeys, there was a significant difference between the Auburn and B.U.T. Premium turkeys . We noticed no significant difference in body injuries per breed between beak-trimmed and non-beak-trimmed turkeys with blunting disk . The Auburn turkeys had significantly fewer snood injuries (22.0%) than the other two breeds  because of the small number of affected turkeys. For the variable breast button, we detected significantly  fewer AuThe average gaseous ammonia content per phase was between 0 parts per million (ppm) in Phases 1 and 2 and 2.25 ppm in Phase 6. The average light intensity in the barn was 22 lx. The temperature, measured at the four predetermined locations in the barn , was on At slaughter in Ampfing, Germany, rejections in each group were handled separately, so that total weight of the rejected parts and the reason for rejection could be recorded. The total rejections showed tendencies between the three breeds , but no clear trend considering beak-trimmed and non-beak-trimmed birds. Comparing total rejections of beak-trimmed birds (32.0 kg = 53%) with those of non-beak-trimmed birds with integrated blunting disk (28.8 kg = 47%) revealed a slight tendency in favor of non-beak-trimmed birds. Whole wings and wing tips were rejected most frequently (V1: 23 wings > V2: 18 wings > V3 and V4: 7 wings > V6: 3 wings > V5: 2 wings). In most cases, hematomas on the wings were the reason for rejection. Hematomas also had to be excised from other body parts. In some cases, the lower legs were cut off. Breast buttons (BBu) were another frequent reason for rejection (V3: 15 BBu > V2 and V4: 13 BBu > V1: 4 BBu > V6: 1 BBu > V5: 0 BBu).The beak measurements of the slaughtered turkeys after Phases 5 and 6 are shown in The weight of the turkeys after Phase 6 (weighing on the 137th DOL) differed according to breed but not according to beak condition (beak-trimmed vs. non-beak-trimmed with the blunting disk). These results are contrary to those of our preliminary study, in which the turkeys with integrated screed grinding wheel after Phase 6 weighed significantly less than the beak-trimmed turkeys without blunting . In the 2) than for the other two breeds . Kulke et al. [2 vs. 58 kg/m2) in non-beak-trimmed B.U.T. 6 male turkeys and found no differences considering mortality or stocking density. They only found a significant difference in the final fattening weight, with the birds fattened at lower stocking density showing a higher body weight at the end of fattening.The mortality rate of 0.7% during rearing and 4.2% during fattening in the present study can be considered low for male turkeys. In the preceding study with B.U.T 6 turkeys , in whice et al.  investigFurthermore, we noticed that the Auburn turkeys had fewer body injuries, snood injuries, breast buttons, and tail feather injuries than the turkeys of the other two breeds. Bergmann  also fouIn the present study, we found no significant difference in the assessed body injuries per breed between the non-beak-trimmed turkeys with blunting disk and the beak-trimmed turkeys. This finding, along with our results on beak morphology, suggests that the 30-grit grinding wheels in the feed pans caused a beak abrasion that was similarly effective in preventing pecking injuries as the infrared beak trimming. Several studies showed that beak-trimmed turkeys have fewer injuries and less feather loss than non-beak-trimmed turkeys ,15,17. FIn the beak evaluation, we noticed for both the beak scoring and the measurements that the B.U.T. Premium turkeys tended to show the strongest abrasion, although beak abrasion also occurred in the other two breeds. Fiks-van Niekerk and Elson , who invRegarding the assessed climate parameters, the gaseous ammonia content in the barn air was below 5 ppm during all measurements and thus always below the maximum permitted limit of 20 ppm . The ligRegarding the effectivity of the blunting disk, we could prove beak abrasion in all three breeds. Although B.U.T. Premium turkeys had the shortest upper-beak overlap by trend, they did not have fewer injuries than the Auburn or the B.U.T. 6 turkeys. Thus, considering the assessed variables , the blunting method for a natural beak abrasion smoothing examined in this study was just as effective as beak trimming with the described infrared beam setting. Furthermore, the Auburn breed showed fewer injuries, plumage damages and rejections, but had poorer foot pad health than the turkeys of the other two breeds. Thus, we can conclude that the fattening of a robust turkey strain shows benefits in terms of animal welfare. Moreover, after correct application of the blunting disk, no pain, suffering, or damages due to this method were detected, neither macroscopically nor histologically. However, these findings are based on the investigation of one fattening phase with a limited number of animals and replicates. In summary, this blunting method represents an improvement in animal welfare and should be further investigated in practice.


Hearing impairment is a frequent human sensory impairment. It was estimated that over 50% of those aged >75 years experience hearing impairment in the United States. Several hearing impairment–related factors are detectable through screening; thus, further deterioration can be avoided. Early identification of hearing impairment is the key to effective management. However, hearing screening resources are scarce or inaccessible, underlining the importance of developing user-friendly mobile health care systems for universal hearing screening. Mobile health  applications (apps) act as platforms for personalized hearing screening to evaluate an individual’s risk of developing hearing impairment. We aimed to evaluate and compare the accuracy of smartphone-based air conduction and bone conduction audiometry self-tests with that of standard air conduction and bone conduction pure-tone audiometry tests. Moreover, we evaluated the use of smartphone-based air conduction and bone conduction audiometry self-tests in conductive hearing loss diagnosis. We recruited 103 patients (206 ears) from an otology clinic. All patients were aged ≥20 years. Patients who were diagnosed with active otorrhea was excluded. Moderate hearing impairment was defined as hearing loss with mean hearing thresholds >40 dB. All patients underwent four hearing tests performed by a board-certified audiologist: a smartphone-based air conduction audiometry self-test, smartphone-based bone conduction audiometry self-test, standard air-conduction pure-tone audiometry, and standard bone conduction pure-tone audiometry. We compared and analyzed the results of the smartphone-based air conduction and bone conduction audiometry self-tests with those of the standard air conduction and bone conduction pure-tone audiometry tests. The sensitivity of the smartphone-based air conduction audiometry self-test was 0.80  and its specificity was 0.84 (95% CI = 0.76–0.90), respectively. The sensitivity of the smartphone-based bone conduction audiometry self-test was 0.64 (95% CI = 0.53–0.75) and its specificity was 0.71 (95% CI = 0.62–0.78). Among all the ears, 24 were diagnosed with conductive hearing loss. The smartphone-based audiometry self-tests correctly diagnosed conductive hearing loss in 17 of those ears. The personalized smartphone-based audiometry self-tests correctly diagnosed hearing loss with high sensitivity and high specificity, and they can be a reliable screening test to rule out moderate hearing impairment among the population. It provided patients with moderate hearing impairment with personalized strategies for symptomatic control and facilitated individual case management for medical practitioners. Hearing impairment is among the most prevalent sensory impairments in human beings. As documented in various studies, the worldwide prevalence of hearing loss is approximately between 4.0% and 18.1% ,3,4,5. DNevertheless, numerous countries experience practical challenges in preventing and rehabilitating hearing impairment. The first obstacle is the limited medical resources failing to meet patient demands. The diagnostic benchmark for hearing impairment is the standard audiogram. Nevertheless, financial or geographic restrictions can impede prompt audiogram testing . AdvanceAccordingly, we attempted to develop a cost-effective hearing examination for evaluating an individual’s hearing condition. Smartphone-based hearing tests have been developed as screening and assessment tools to recognize patients with hearing deficits. Hence, our aim was to assess the efficacy of smartphone-based audiometry tests in screening patients with moderate hearing impairment and to validate these tests against standard pure-tone audiometry. We also evaluated and compared the accuracy of smartphone-based air conduction and bone conduction audiometry self-tests with their corresponding standard pure-tone audiometry tests. Secondarily, we aimed to evaluate the effectiveness of smartphone-based air conduction and bone conduction audiometry self-tests for diagnosing conductive hearing loss.In total, 103 participants  who were aged older than 20 years were recruited to the otology outpatient department of Kaohsiung Veterans General Hospital and Shuang Ho Hospital. Patients with existing otorrhea and cognitive impairment were excluded from recruitment. For both ears of each participant, four hearing examinations were performed randomly: a smartphone-based air conduction audiometry self-test, smartphone-based bone conduction audiometry self-test, standard air-conduction pure-tone audiometry, and standard bone conduction pure-tone audiometry. These tests were performed by a board-certificated audiologist in randomized order and in a soundproof room with an average A-weighted ambient noise level of 35 dB during each visit. Informed consent was obtained from all patients.The four hearing tests for all participants were conducted by a board-certificated audiologist in a soundproof room randomly. The four tests were arranged in random order and there was no time lag between tests. We did not calibrate because we aimed to stick to the reality in which calibration may not be readily available even if the patients use the same smartphone and headphone. Mean hearing thresholds were defined at 500, 1000, 2000, and 4000 Hz. For hearing loss with mean hearing thresholds >40 dB, moderate hearing impairment was impressed. We applied the 2 × 2 tables to summarize the statistics in our study. The results were collected for further evaluation and statistical analysis.Audiometry self-tests were completed using an iPhone . An application named uHear , through which patients can test their air conduction pure-tone hearing sensitivity, was downloaded to the phone through the iTunes App Store. uHear employs a “10-dB down and 5-dB up’’ principle ,19, and This self-test was also completed with an iPhone . The uHear application  was downloaded from the iTunes App Store. The procedure of this smartphone-based bone conduction audiometry self-test was the same as that of the air conduction variant. The results of the hearing sensitivity test were visualized in a typical audiogram format after test completion. Furthermore, the smartphone-based bone conduction audiometry self-test was conducted in a soundproof room whose average ambient noise level was less than 35 dB HL. AfterShokz Sportz 2 bone conduction headphones  were used for all participants. This smartphone-based self-test was completed by each participant in a soundproof room. Finally, we put the results of this smartphone-based bone conduction audiometry self-test and those of standard bone conduction pure-tone audiometry tests into comparison.A Grason-Stadler GSI-61 clinical audiometer  was used to perform the standard pure-tone audiometry test. Telephonics TDH-50P audiometric headphones  were used to perform the test. All clinical audiometers and accessory devices met 1996 American National Standards Institute S3.6 guidelines . The tesThe Grason-Stadler GSI-61 clinical audiometer as well as a B-71 bone conductor  were used to perform the standard bone conduction pure-tone audiometry test. All clinical audiometers and accessories met the 1996 American National Standards Institute S3.6 guidelines . The tesIn total, 103 patients  were examined in our study. The mean age of the patients was 55.23 ± 6.14 years. Of these patients, 69 were men and 36 were women. Detailed patient demographics are summarized in As indicated in The results of the smartphone-based bone conduction audiometry self-test were compared with that of the standard bone conduction pure-tone audiometry test, as presented in Conductive hearing loss is defined as an air–bone gap of at least 20 dB compared with a normal bone conduction hearing threshold. Among the 206 ears, 24 were diagnosed with conductive hearing loss by using the standard air conduction and bone conduction pure-tone audiometry tests. The smartphone-based air conduction and bone conduction audiometry self-tests correctly diagnosed 17 of those 24 ears with conductive hearing loss.Masalski et al. reported a 15.6% worldwide prevalence of hearing impairment on the basis of 116,733 hearing tests performed by Android users on mobile devices ,18,21. PIn our study, we compared results from a smartphone-based air conduction audiometry self-test and those from a standard air conduction pure-tone audiometry test. Among the 88 ears determined as having moderate hearing impairment (PTA > 40 dB HL) by the standard air conduction pure-tone audiometry test, 71 recorded a PTA of >40 dB HL in the smartphone-based air conduction audiometry self-test, indicating an 80% sensitivity. Moreover, among the 118 ears without moderate hearing impairment (PTA ≤ 40 dB HL) documented in the standard air conduction pure-tone audiometry test, 99 registered a PTA of ≤40 dB in the smartphone-based air conduction audiometry self-test, demonstrating 84% specificity. These findings coincide with those of previous studies. Li et al. observed that the sensitivity and specificity of smartphone-based self-tests performed by older (>65 years) adults were 92% and 76%, respectively . Sara etOur pioneering study also correlated the accuracy of the smartphone-based bone conduction audiometry self-test and that of the standard bone conduction pure-tone audiometry test. Among the 76 ears with moderate hearing impairment (PTA > 40 dB HL) documented in the standard bone conduction pure-tone audiometry test, 49 registered a PTA of >40 dB HL in the smartphone-based bone conduction audiometry self-test, indicating a sensitivity of 64%. Furthermore, among the 130 ears deemed not to have moderate hearing impairment (PTA ≤ 40 dB HL) in the standard bone conduction pure-tone audiometry test, 92 recorded a PTA of ≤40 dB in the smartphone-based bone conduction audiometry self-test, suggesting a 71% specificity. Previous studies have mostly focused on smartphone-based air conduction audiometry self-test accuracy. By contrast, our study is the first to use AfterShokz Sportz 2 bone conduction headphones to perform experiments on the accuracy of smartphone-based bone conduction audiometry. Moreover, by comparing the results of standard air conduction pure-tone audiometry with those of standard bone conduction pure-tone audiometry, we observed that standard air conduction pure-tone audiometry possessed higher sensitivity as well as higher specificity. This difference was due to the sound leakage engendered by the AfterShokz Sportz 2 bone conduction headphones used in the smartphone-based bone conduction audiometry test. To resolve this problem, we thoroughly compared the AfterShokz Sportz 2 bone conduction headphones with the Sennheiser hd201 headphones, discovering that the Sennheiser hd201 headphones fit snugly in the ear canals. This may explain the higher sensitivity and specificity observed in the smartphone-based air conduction audiometry self-test.Conductive hearing loss is defined as an air–bone gap of at least 20 dB compared with a normal bone conduction hearing threshold. Among the 206 ears, 24 were diagnosed with conductive hearing loss in the standard air conduction pure-tone audiometry test and standard bone conduction pure-tone audiometry test. Our results indicate that smartphone-based air conduction and bone conduction audiometry self-tests correctly diagnosed 17 of these 24 ears with conductive hearing loss.The reason why this study is important and novel is that we aimed to develop an accurate method to detect hearing impairment with the use of commonly available portable mobile devices, so that early diagnosis and prompt treatment of hearing impairment can be achieved.This study has several limitations. First, the standard air conduction and bone conduction pure-tone audiometry protocol requires audiologists to apply masking, which aims to acoustically separate the two ears. However, when conducting the smartphone-based air conduction and bone conduction audiometry self-tests, we did not apply masking noise. During the hearing tests, the contralateral ear may hear a tone presented in the tested ear. This phenomenon is called crossover, and it occurs when an air-conducted signal is intense enough to cause the skull to vibrate. Sound is transmitted through bone conduction, allowing the patient to hear the tone in the nontested ear. In future studies, changes should be introduced to the application to prevent improper reaction to the masking noise.Second, we used AfterShokz headphones in our study because it is one of the most common bone conduction earphones available in Taiwan. We could hardly find mastoid bone-conduction earphones. Despite the fact that the AfterShokz headphone has its bone conduction through the temporomandibular joint, we chose it as the experimental device because of its widespread popularity in the real world. We did not calibrate because we aimed to stick to the reality in which calibration may not be readily available even if the patients use the same smartphone and headphone. We performed the experiment in a soundproof booth in order to minimize the uncontrollable sound effect of the surrounding environment. We aimed to investigate the efficacy of the smartphone-based air audiometry self-test under the circumstance with standardized controllable factors. Testing in more realistic scenarios will be a direction of further research.We chose mobile phones instead of laptops because of their higher coverage in many developing countries. Thanks to the production of domestic mobile phone models, mobile phones have become more affordable for people’s daily communication. The overall cost of our study was 330 US dollars, consisting of 200 US dollars for the mobile phone, 50 US dollars for the air conduction earphone, and 80 US dollars for the bone conduction earphone.Besides, in order to minimize possible errors, all participants underwent the experiment under an audiologist’s guidance. It is agreed that if patients were asked to carry out the experimental procedures by themselves, many of them may fail to perform the test, which in turn causes larger procedural variation and affects the participant inclusion of our study. We included the audiologist’s instruction because many people in Taiwan are not familiar with English and it would be difficult for them to carry out the audiological tests on themselves. In order to deal with these unanswered questions, we hope to carry out further investigation in the future. We wish to perform further studies without the guidance of an audiologist.Both smartphone-based air conduction and bone conduction audiometry self-tests demonstrated high sensitivity and high specificity in diagnosing moderate hearing impairment. The mobile health application was a user-friendly personalized mHealth application, which aids precise evaluation and early intervention of moderate hearing impairment. Compared with a standard pure-tone audiometry test, the smartphone-based air conduction and bone conduction audiometry self-tests proved capable of detecting conductive hearing loss with reliable sensitivity and specificity. In conclusion, it is a reliable hearing screening tool but can only be achieved if hardware is correctly calibrated or at least standardized and the examination is carried out in a quiet environment. We will refine the mobile health application based on users’ experience and the collected dataWe thank all participants and their families for supporting the study.


Oncomodulin (OCM) is a Ca2+ binding protein (CaBP) preferentially expressed in outer hair cells (OHCs) of the cochlea and few other specialized cell types. Here, we expand on our previous reports and show that OCM delays hearing loss in mice of two different genetic backgrounds: CBA/CaJ and C57Bl/6J. In both backgrounds, genetic disruption of Ocm leads to early progressive hearing loss as measured by auditory brainstem response (ABR) and distortion product otoacoustic emission (DPOAE). In both strains, loss of Ocm reduced hearing across lifetime (hearing span) by more than 50% relative to wild type (WT). Even though the two WT strains have very different hearing spans, OCM plays a considerable and similar role within their genetic environment to regulate hearing function. The accelerated age-related hearing loss (ARHL) of the Ocm KO illustrates the importance of Ca2+ signaling in maintaining hearing health. Manipulation of OCM and Ca2+ signaling may reveal important clues to the systems of function/dysfunction that lead to ARHL.Ca Althougronments . Inner hronments . During ronments . The losronments . Therefo2+ regulation . While certain CaBPs such as alpha-parvalbumin (αPV) are down regulated, oncomodulin (OCM), a member of the parvalbumin family, is upregulated in mouse OHCs soon after birth (P2-P3), supplanting other CaBPs as at the dominant CaBP  mouse and C57Bl/6J (C57) mouse, are frequently used as models for late- and early-onset ARHL, respectively . The CBA2+ regulation may play in hearing function, we used the Ocm KO mouse model to explore the link between OCM and ARHL. The C57 strain is known to potentiate hearing loss of other Ca2+-regulatory gene mutations  and KO mice of both backgrounds. In both mouse strains, the Ocm KO allele resulted in an early progressive hearing loss that was at least 50% of the normal hearing span, and a loss of efferent terminals and OHCs. Also in both strains, aged WT mice show alterations in the localization of OCM that coincide with the loss of OHCs and efferent terminals. This study suggests that OCM may indeed play a role in ARHL associated with OHC dysfunction.Given the unique expression profile of OCM in the cochlea, and the role that Cautations . Since C–/–Ocm (Ocm KO) mice were generated from spontaneous germline transmission of the KO allele from the parental line, Cre;Ocmflox/floxActb on the C57Bl/6J background . Homozygous CBA Ocm KO and C57 Ocm KO and WT littermates from each sex were used for all experiments.All experiments were done in compliance with National Institutes of Health and institutional animal care guidelines and were approved by the Institutional Animal Care and Use Committee of Baylor University. ckground . The orilleagues . The CdhCdh23ahl . ConfirmTissues were incubated with primary antibody overnight at 37°C. Primary antibodies used: goat anti-OCM, , rabbit anti-prestin , goat ai.p.). Acoustic stimuli were delivered using a custom acoustic assembly previously described by For measurement of auditory brainstem responses (ABRs) and distortion product otoacoustic emissions (DPOAEs), adult mice were anesthetized with xylazine  and ketamine  were perfused transcardially with 4% paraformaldehyde (PFA) in 0.1M phosphate buffer (PBS) followed by PBS wash. After removal, cochleae scalae were flushed with PFA then rotated in fixative overnight at 4°C. Cochleae were decalcified in 0.1M EDTA for 3–5 days at 4°C with rotation. Cochleae were prepared as cochlear whole mounts of approximately 6 pieces of sensory epithelium or as 100 μm mid-modiolar sections embedded in gelatin-agarose solution . Cochlea2 The plugin superimposed frequency correlates on the microdissected spiral image by application of the cochlear frequency map for mice. Organ of Corti was imaged at the 8, 16, and 32 kHz regions with the LSM800 confocal microscope using a 63x oil immersion objective (N.A. 1.4). At each of the desired locations, 3 adjacent microscopic fields (9–12 IHCs per field) were imaged with a 4-channel z-stack spanning the height of the hair cells to capture the stereocilia, nuclei and synaptic junctions. Hair cells were only counted if they had a nucleus or hair bundle/cuticular plate present. ChAT-labeled efferent clusters of terminal swellings were counted in equally spaced distance regions using a LSM5 confocal microscope using a 40x oil immersion objective (N.A. 1.2). In each cochlear location, we acquired two adjacent z-stacks , taking care to span the entire region containing ChAT-positive terminals along the z-axis. Because of the difficulty counting ChAT-labeled terminals in older animals, we resorted to counts only of clusters of labeled efferent terminals. Counts were performed by a blinded observer.Low-power images of each microdissected piece of surface preparations were obtained with a 10x air objective (N.A. 0.3) on a LSM800 confocal microscope. Cytocochleograms were constructed from these images by tracing the cochlear spiral and superimposing hash marks using a custom ImageJ Measure Line plugin from Eaton-Peabody.For OCM subcellular distribution, quantification was performed in ImageJ using a region of interest (ROI) from the nucleus compared with a similar ROI in the cytoplasm. After background removal, cytoplasmic OCM fluorescence intensity was divided by nuclear OCM fluorescence intensity. 2-5 cochlea from different animals were analyzed per condition.p < 0.05), followed by Dunnett’s multiple comparisons test. Mann-Whitney U-test was applied when normal distribution cannot be assumed. Mean values are quoted in text and figures as means ± SEM (DPOAE and ABR measurement) and means ± SD (missing OHC/ChAT counting and OCM distribution). p < 0.05 was selected as the criterion for statistical significance.All statistical analysis were performed in Prism (v9.1x GraphPad Software). All data were tested for homogeneity of variance. For multiple comparisons, Brown-Forsythe and Welch ANOVA test was applied when SDs are significantly different . To test hearing function, ABR waveforms of C57 KO . In contrast, WT animals demonstrate similar ABR thresholds at low and middle frequencies from 2 to 7 mo, and only show substantial threshold shifts in the high frequencies at 5 - 7 mo and middle frequencies at a year .We previously showed that targeted deletion of 3 – 4 mo . We wantf C57 KO  and CBA f C57 KO  mice at f C57 KO . Ocm KOsC57 mice  and 7 moC57 mice , whereaso of age . Tong eto of age . WT and o of age . In the t a year . At 16 kOcm KO in C57Bl/6J mice did not change the original hearing loss phenotype described by p < 0.0001; Dunnett’s test: p = 0.009 for 1 – 3 mo, p = 0.002 for 3 – 6 mo). In the C57 KO between 3 and 6 mo, we observed sizable threshold shifts in all but the lowest frequencies (8 - 32 kHz), while in the WT, sizable threshold shifts were observed between 5 and 16 mo . Since OCM is predominantly expressed in OHC of the organ of Corti at these ages, these results confirm that OHC defects are primarily responsible for the differences in ABR potentials between the Ocm KO early onset hearing loss and the WT.To confirm that germ-line transmission of the nd 16 mo . The larp < 0.0001; Dunnett’s post-test: p < 0.0001 for 2 – 7 mo, p < 0.0001 for 2 – 12 mo). In contrast, WT mice maintained healthy threshold levels at least up to 16 mo of age and produced measurable DPOAE threshold responses in most frequencies by 28 mo . Interestingly, young adult KO mice (2 mo) show enhanced DPOAE input-output functions compared with WT animals suggesting enhanced electromotility at least for higher frequencies. We compared DPOAE input-output functions at 32 kHz across ages (p < 0.05).At 16 kHz in CBA mice, only DPOAE thresholds comparisons between 2 and 28 mo were significantly different  of mice regardless of genetic background . This phenomenon is more extreme in basal regions and at older ages . Both KO OHC and efferent loss appear similar to recent data collected from young and old human temporal bones  efferent axons protects OHCs from excessive noise and Camotility . Loss ofmotility . Therefomotility  and mid-motility #. At 30 motility . The KOsmotility ∗#. Both al bones . The WT In C57 mice, the pattern of OHC and efferent loss was more pronounced. The WT apical OHC and efferent loss was evident as early as 5 mo and by 15 mo both apical and basal OHCs and efferent clusters were mostly absent . In the We analyzed the loss of OHCs and ChAT-labeled efferent connections in relation to specific DPOAE frequency regions in CBA KO animals . We co-lOcm KO mouse using C57Bl/6J and then backcrossed the Ocm KO onto CBA/CaJ. Compared to their WT controls, both Ocm KO strains demonstrated an earlier progressive elevation of hearing thresholds, loss of efferent synaptic contacts, and OHC loss. Our results in the Ocm KO suggest that alterations in Ca2+ signaling lead to OHC dysfunction before loss of efferent synaptic contacts and/or OHC loss. Although we do not have direct evidence, our results suggest that changes in the presence of efferent terminals may occur prior to OHC loss. The WT and Ocm KO mice share common aging characteristics despite differences in their temporal progression of hearing loss. Ocm KO animals across frequencies. Both aged WT and young KO show OHCs and ChAT labeled efferent connections are lost with age. Importantly, even when ABR and DPOAE thresholds are highly elevated or not measurable across frequencies, the majority of OHCs and efferent terminals are still present in most frequency regions in both aged WT and younger Ocm KO animals, suggesting compromised OHC electromotility function. At least in OHCs, it is possible that disturbances in Ca2+ signaling caused by the removal of OCM lead to the early activation of a general aging process. The absence of OCM in OHCs appears to accelerate ARHL and mimics human presbycusis, which typically displays loss of nerve fibers and hair cells at apex and base  show no hearing loss phenotype  progressive elevation of DPOAE thresholds, (2) loss of OHC efferent terminals, and (3) OHC loss. In Ocm KOs on a CBA background, our data show a progressive elevation of DPOAE thresholds over 12 mo. In contrast, WT hearing thresholds and response magnitudes change little from 2 to 12 mo. Similar to our observations in the C57 strain, young (2 mo) CBA Ocm KO mice had DPOAE thresholds nearly identical to WT controls at lower frequencies and somewhat enhanced at higher frequencies. In older CBA Ocm KO mice, maximum DPOAE threshold shifts occurred at frequencies 16 kHz and higher, which is within the region of greatest hearing sensitivity in these animals. Ocm KO mice had significantly higher thresholds at all measured frequencies between 5 and 7 mo. At 12 mo, DPOAE thresholds were only slightly elevated in WT control mice and were not measurable in the Ocm KO. In the present study, WT CBA mice had measurable DPOAEs up to 28 mo and we did not find significant ARHL until after 28 mo. These results suggest that Ocm deletion in CBA mice leads to an accelerated ARHL phenotype that is faster than the WT C57, but more delayed than in C57 KO mice. Although the specific temporal pattern of hearing loss differs, the results were similar to our previous report and validate that OCM expression is critical for either the maintenance of cochlear function and/or protecting OHCs from damage as previously hypothesized , which store and release Ca2+ as well as alter ATP utilization. Although it is possible the overall mRNA expression of Ocm and Slc26a5 (prestin) in the cochlea decreases with age as reported by 2+ activity. It seems one of our challenges will be separating general features of aging in OHCs from those that lead to ARHL.In ated DPs . However and Ocm . Most, i and Ocm . However and Ocm , most li2+ entry through α9α10 nicotinic ACh receptors (α9α10 nAChRs). The Ca2+ influx activates a hyperpolarizing current through Ca2+-activated small conductance K+ (SK2) channels on the OHC and causes OHCs to elongate , loss of efferent synapses, OHC loss, and hearing loss. Likely, there are multiple avenues for disrupting this aging process, which leads to the differences in ARHL progression that we see across animal models. By interfering with Ca2+ signaling, which is fundamental to many intracellular processes, we were able to expose the process and enhance its destabilization.By comparing loss of OCM in two mouse strains with contrasting temporal patterns of ARHL, we have demonstrated that OCM and CaThe raw data supporting the conclusions of this article will be made available by the authors, without undue reservation.The animal study was reviewed and approved by the Baylor University Institutional Animal Care and Use Committee.LC and DS wrote the manuscript. LC, AH, KM, AC, AL, and PS performed the tissue preparation, immunostaining, and imaging. LC, DS, AH, YY, AC, and AL contributed to ABR and DPOAE analysis. All authors discussed and edited the manuscript. All authors contributed to the article and approved the submitted version.The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest.All claims expressed in this article are solely those of the authors and do not necessarily represent those of their affiliated organizations, or those of the publisher, the editors and the reviewers. Any product that may be evaluated in this article, or claim that may be made by its manufacturer, is not guaranteed or endorsed by the publisher.


The retina of CLN5−/− sheep shows progressive atrophy of the outer retinal layers, widespread gliosis, and accumulation of lysosomal storage in retinal ganglion cells late in disease. In contrast, CLN6−/− retina shows significant atrophy of all retinal layers, progressive gliosis, and earlier accumulation of lysosomal storage. This study has highlighted the differential vulnerability of retinal layers and the time course of retinal atrophy in two distinct models of NCL disease. This data will be valuable in determining potential targets for ocular therapies and the optimal timing of these therapies for protection from retinal dysfunction and degeneration in NCL.Neuronal ceroid lipofuscinoses  are a group of inherited neurodegenerative diseases with a common set of symptoms including cognitive and motor decline and vision loss. Naturally occurring sheep models of CLN5 and CLN6 disease display the key clinical features of NCL, including a progressive loss of vision. We assessed retinal histology, astrogliosis, and lysosomal storage accumulation in CLN5 affected (CLN5 Neuronal ceroid lipofuscinoses  are a group of recessively inherited lysosomal storage disorders. NCL presents primarily as a progressive neurodegenerative disease in children and is ultimately fatal. There are currently 13 known variants of NCL, designated CLN1-8, and CLN10-14. Common symptoms across most variants of NCL include cognitive and motor regression, seizures, and progressive loss of vision.1. The diseased retina has been observed to be no more than a glial scar at post-mortem, however the onset and progression of retinal cell loss in human patients is still unclear1. Several animal models of NCL, including rodents, canines, and livestock demonstrate similar visual deficits and retinal pathology to NCL patients, making them valuable tools in studying the retinal component of disease7. Naturally occurring models of NCL exist in sheep in New Zealand; CLN5 disease in Borderdale sheep, and CLN6 disease in South Hampshire sheep9. These models replicate the primary symptomatic and neurological profiles of NCL, including a progressive loss of vision12.Vision loss in NCL is thought to be due to cell death in both the visual cortex and in the retina. Cortical atrophy and neuronal cell loss is most pronounced in the occipital lobe of patients at end-stage diseaseMany of the studies of retinal tissue from NCL patients or models have been done at end stage disease, therefore a detailed analysis of the onset and progression of retinal dystrophy in NCL is still lacking. Understanding the differential vulnerability of retinal cells at distinct disease stages will be useful in determining the main target cells and optimal timing of potential therapeutics. This comprehensive natural history study assesses retinal histology and lysosomal storage burden in two different sheep models of NCL between pre-symptomatic and end-stage disease.−/− sheep eye following a similar trajectory. The transverse diameter of the healthy sheep eye was 27.9 ± 0.8 mm at 5 months of age and had grown to 29.1 ± 0.1 mm by 18 months of age. In the CLN5−/− sheep eye the transverse diameter was similar to healthy sheep between 5 and 10 months of age, then began to diverge and was significantly lower (28.2 ± 0.16 mm) than healthy sheep at 18 months of age  and declines steadily to 161 ± 2.2 µm at 18 months of age  is significantly thicker compared to CLN5−/− sheep  compared to controls (24%), and lower proportions of ONL (7%) and IS/OS (7%) compared to controls  at 3 months of age, leading to a significantly higher average number of rows in CLN5−/− sheep compared to controls at this age. However, at 6, 12, and 18 months the number of ONL rows was significantly lower in CLN5−/− sheep compared to controls, with ONL rows remaining stable at 6.9 ± 0.05 rows in controls over time but declining to an average of 1.7 ± 0.06 rows in CLN5−/− sheep by 18 months of age.The number of rows of nuclei in the ONL was quantified in the central retina of control and CLN5age Fig. c. One CL−/− retina compared to controls  signal in the central retina was studied in conjunction with lysosomal protein LAMP1 to determine the extent of lysosomal storage burden in the CLN5ols Fig. . In the MP1 Fig. . By 18 m−/− animals however they were morphologically irregular in shape and their processes had a more disorganised appearance compared to the control NFL. From 6 months of age, GFAP immunoreactivity had spread to the outer retinal layers of the diseased eye, in the form of vertically elongated Müller cell processes, with some Müller cell bodies evident by 18 months of age  than control (117 ± 1.5 µm) peripheral retina at 18 months of age  were significantly thicker in CLN6−/− retina, AF signal was evident from 3 months of age but did not co-localise with LAMP1 at this age. By 6 months of age there was faint AF signal in LAMP1-positive ganglion cell bodies in the CLN6−/− retina, which became stronger and more distinct at 12 and 18 months of age  were selected to represent four distinct clinical disease stages, being pre-symptomatic, early symptomatic, symptomatic and end-stage disease respectively.16. In many of these diseases the early increase in retinal thickness has been attributed to inflammatory edema which occurs in response to photoreceptor cell stress prior to retinal atrophy16. Although early upregulation of GFAP was not observed in affected sheep retina, it would be useful in future to assess these retina for other signs of inflammation such as microglial infiltration, or upregulation of Müller cell specific markers or inflammatory cytokines.Total retinal thickness was assessed in both the central and peripheral retina, with the central retina showing an earlier and more distinct difference between control and affected sheep. In both breeds the central retina was thicker in affected animals compared to controls at 3 months of age. This may be due to early compensatory mechanisms or inflammatory edema in the retina of affected animals, however requires more investigation. Compensatory mechanisms may include upregulated mRNA or protein expression of neuroprotective markers and could be assessed by molecular or histological analysis of pre-symptomatic sheep retina. Although early increases in retinal thickness have not been observed in other animal models of NCL, they have been observed in several degenerative diseases including age-related macular degeneration, optic neuritis, diabetic retinopathy, and leber congenital amaurosis−/− sheep from 6 months onwards, however growth was slower in control South Hampshire sheep. Despite initial differences in peripheral retina thickness between control and affected sheep of both breeds, the thicknesses were similar from 6 months of age until a significant drop in thickness in CLN5−/− and CLN6−/− sheep at end-stage disease. While total retinal thickness is informative for tracking global changes in the retina, it is important to consider the structure and function of individual layers of the retina and how these change over the course of disease. This is particularly pertinent in CLN5−/− retina at end stage disease where total thickness is comparable to controls, however almost 40% of this total thickness is taken up by the NFL. Meanwhile there is severe shrinkage of the ONL and IS/OS, which is more likely to be the underlying cause of retinal dysfunction. The cause and physiological consequences of this increasing NFL thickness in affected animals is unknown. In CLN5−/− sheep it is primarily the outer retinal layers  which show significant degeneration over the disease course. While inner retina  thicknesses do fluctuate over time, they are more comparable to control at end stage disease. In contrast, all retinal layers in CLN6−/− sheep significantly degenerate over the course of the disease, except for the RPE.There was early growth of the central retina in control Borderdale sheep leading to thicker retina in control compared to CLN5−/− sheep compared to CLN5−/− sheep. Minor amounts are observed in the CLN6−/− retina at 6 months of age, which then progresses until ganglion cell bodies appear full of storage by end stage disease. This is in keeping with early studies of the CLN6−/− sheep retina, which showed atrophy of photoreceptor cell bodies and inner and outer segments, and accumulation of autofluorescent storage material primarily in ganglion cells11.In both breeds, lysosomal storage occurs primarily in ganglion cell bodies, however it appears earlier in the retina of CLN618. However, in affected sheep NFL resident astrocytes had an altered morphology and, from 6 months of age, GFAP-positive cells were evident throughout the retina. Müller cells typically do not express GFAP, but upregulate its expression in response to pathological processes in the retina19. GFAP-expressing Müller cells were evident in the outer retina of affected animals from 6 months of age, indicating the initiation of reactive gliosis. Early and progressive increases in GFAP immunoreactivity and activation of Müller cells has also been observed in the retina of several mouse models of NCLs23.In the control sheep retina at all ages studied, GFAP immunoreactivity was primarily confined to horizontally elongated cell bodies and processes in the NFL, reminiscent of quiescent astrocytes commonly found in the healthy retina12. This coincides with the age when clinical signs of visual deterioration, such as reduced menace response and pupillary light reflex, also begin to occur in affected sheep24. By 15 to 17 months of age ERG responses are abolished in the majority of affected sheep and they can no longer navigate through a maze24. The retinal pathology presented in this study aligns with these clinical features as the number of ONL rows, indicative of photoreceptor cell body numbers, show significant decline from 6 months of age and diminish over the disease course.A significant reduction in ERG a- and b-waves in CLN5 and CLN6 affected sheep occurs from 9 to 11 months of age, suggesting dysfunction of photoreceptor and bipolar cells26.Post-mortem data from retina tissue of NCL patients is rare, however what is known about the retina of CLN5 and CLN6 patients is summarised in Table 31, due to its similarities in globe size and retinal structure to humans. There are several different potential approaches for delivering corrective therapies to the retina in NCL, including intravitreal, subretinal, suprachoroidal, periocular, or topical administration. Of these, intravitreal delivery is one of the least invasive as it involves injection of the drug to the vitreous cavity of the eye, but has less target specificity than a subretinal or suprachoroidal delivery route. While the intravitreal route is not specifically targeted to the outer retina and has barriers such as the inner limiting membrane (ILM) to overcome there are several reasons to consider this approach for retinal diseases caused by soluble protein deficiencies. For example, in CLN5 disease if only inner retinal cells were corrected through intravitreal therapy they could secrete functional CLN5 protein to other retinal cells, a mechanism known as cross-correction32. In addition, given the fragility of the diseased retina it is likely the ILM is disrupted and more permeable in diseased eyes, and in fact others have shown better viral vector transduction in diseased eyes compared to wild-type33. Intravitreal drug delivery is commonplace in clinical settings for a range of ocular diseases36, and retinal dysfunction and pathology was successfully ameliorated up to 18 months of age following a single intravitreal injection of CLN5 gene therapy in CLN5−/− sheep37. Biotechnology company Neurogene Inc has recently announced the planned initiation of a Phase I/II clinical trial for CLN5 disease utilising both intracerebroventricular and intravitreal delivery routes38 and a clinical trial is underway for the intravitreal delivery of the CLN2 enzyme replacement therapy Brineura .The sheep eye is increasingly being recognised as a good model for studying human retinal disease and for testing ocular therapies−/− sheep showed some attenuation of pathology but no rescue of visual function37. A combination of approaches such as suprachoroidal and intravitreal may be appropriate in this case as suprachoroidal would target outer retinal cells with a lower risk of retinal detachment, and the intravitreal route would target inner retinal cells. The general consensus on timing of treatment for degenerative diseases is often ‘the earlier the better’. Our results in NCL sheep have reaffirmed this, as atrophy of retinal layers and accumulation of lysosomal storage start becoming evident by 6 months of age. In addition, early treatment is preferable if using the intravitreal approach as the ILM is still developing and therefore more permeable in younger animals40.The outlook for treating the retinal component of CLN6 disease is more complicated as we have shown that all layers of the retina significantly degenerate in CLN6 affected sheep, and CLN6 is a membrane bound protein meaning cross-correction is unlikely to be of benefit. Indeed, a recent trial of intravitreal gene therapy in CLN6This systematic study of the progression of retinal degeneration in distinct sheep models of CLN5 and CLN6 NCL has highlighted the differential vulnerability of retinal cell types in each disease and the time course of degeneration. In CLN5 sheep, the outer retina is most severely affected, with significant thinning of the outer nuclear layer evident from 6 months of age. Conversely in CLN6 sheep, all retinal layers show significant degeneration from 12 months of age. Lysosomal storage accumulation in retinal ganglion cells was a common feature in both sheep models, which is in keeping with observations of post-mortem human retina. This histological data, in addition to assessment of sheep eye globe dimensions, further validates the use of sheep to study the retinal component of NCL and potential ocular therapies to attenuate retinal dysfunction and degeneration.9 and maintained at the Lincoln University research farm under US National Institutes of Health guidelines for the care and use of animals in research and the NZ Animal Welfare Act (1999). Homozygous affected ewes from both breeds  were sacrificed at 3, 6, 12, and 18 months of age along with age-matched healthy heterozygous flockmates as controls . All experimental protocols were approved by the Lincoln University Animal Ethics Committee. All studies are reported in accordance with the ARRIVE Essential 10 guidelines.Borderdale and South Hampshire sheep were diagnosed at birthSheep were sacrificed by penetrating captive bolt to the cervical spine followed by rapid exsanguination. Eye globes were enucleated at the time of death, fixed in 10% formalin for a minimum of 2 h, and sent to Gribbles Veterinary Pathology, Christchurch, NZ for post-fixation in Bouin’s solution  for 4 h, followed by wax embedding. Retinal paraffin sections were cut at 3 µm, mounted and a subset stained with Hematoxylin and Eosin (H + E) histological stain by Gribbles Veterinary Pathology for analysis of retinal thickness and layer differentiation.20 and sections were coverslipped in BrightMount plus anti-fade mounting medium (Abcam ab103748).Retinal paraffin sections were stained with either lysosome-associated membrane protein 1  or glial fibrillary acidic protein . Sections were de-waxed twice in xylene (5 min) and rehydrated through a graded ethanol series to water. Following re-hydration, sections underwent antigen retrieval in 10 mM Sodium Citrate buffer (pH 6) at 80 °C for 30 min, then were allowed to cool for 20 min. Sections were then washed in TBST  and blocked in 10% Normal Goat Serum (NGS) in TBST for 30 min at room temperature, before overnight incubation in the primary antibody in 10% NGS in TBST at 4 °C. Sections were washed in TBST and then incubated in goat anti-rabbit Alexa Fluor® 594 secondary antibody  10% NGS in TBST, 1 h, in the dark at room temperature. Sections were then washed and incubated in DNA stain DAPI  for 3 min at room temperature. DAPI was washed off in dHAll H + E stained sections were imaged on a Nikon Eclipse 50i microscope  paired to a Nikon Digital Sight DS-U3 camera and NIS-Elements BR software (v. 4.50 Nikon Instruments). Ten total retinal thickness measurements per eye were taken by a blinded researcher from the surface of the nerve fibre layer (NFL) to the base of the retinal pigment epithelium (RPE) in both the central retina (within 5 mm of the optic nerve head) and the peripheral retina (10–20 mm from the optic nerve head). Individual retinal layer thicknesses were calculated from ten measurements per eye taken from the central retina. Immunofluorescent GFAP signal was imaged using a CY3 Brightline 531 excitation/593 emission filter set .https://imagej.nih.gov/ij/).Co-localisation of LAMP1 and autofluorescent signal was assessed using immunofluorescent images captured on a Zeiss 510 laser scanning confocal microscope with Zen 2009 imaging software . Image collection parameters  were optimised for each channel and remained consistent for all sections in each staining run. High magnification Z-stacks were captured using a 40x/NA 1.3 oil objective and post-processing was performed in ImageJ , diseased CLN5−/− (n = 4), healthy CLN6+/− (n = 4) and diseased CLN6−/− (n = 3) sheep obtained at 5, 7, 10, 14 and 18 months of age. Briefly, sheep were scanned in a 3-Tesla Skyra MRI scanner , with a 20-channel head coil. Sheep were sedated by intravenous injection of 0.5 mg/kg live weight diazepam  and 10 mg/kg live weight ketamine  prior to endotracheal intubation and maintenance on inhalation anaesthesia . Sheep were placed in right lateral recumbency on the patient bed for scanning. DICOM files of T1-weighted scans were loaded into 3D Slicer  and measurements were taken in the axial plane image using the ‘create line markup’ tool.Measurements of axial length and transverse diameter of the sheep eye globe were taken from an existing set of MRI images of healthy CLN5www.graphpad.com).All statistical analysis was performed on GraphPad Prism© . Differences between healthy control and NCL affected sheep were assessed using two-tailed unpaired t-tests at each age . Multiple comparisons were corrected for using the Holm-Sidak method. No statistical comparisons across ages were made as different animals were used at each age.Supplementary Information.


The resulting FA phenoxyl radical, after the scavenging of a second •OH and keto-enol tautomerization of the intermediate, produces 5-hydroxyferulic acid (5OHFA). Inhibition of the lipoxygenase (LOX) enzyme, one of the enzymes that catalyse free radical production, by FA and 5OHFA were analysed. Results of molecular docking calculations indicate favourable binding interactions of FA and 5OHFA with the LOX active site. The exergonicity of chelation reactions of the catalytic Fe2+ ion with FA and 5OHFA indicate the potency of these chelators to prevent the formation of •OH radicals via Fenton-like reactions. The inhibition of the prooxidant LOX enzyme could be more relevant mechanism of skin protection against UVA induced oxidative stress than iron chelation and assumed direct scavenging of ROS.Ferulic acid (FA) is used in skin formulations for protection against the damaging actions of the reactive oxygen species (ROS) produced by UVA radiation. Possible underlying protective mechanisms are not fully elucidated. By considering the kinetics of proton-coupled electron transfer (PCET) and radical-radical coupling (RRC) mechanisms, it appears that direct scavenging could be operative, providing that a high local concentration of FA is present at the place of  The main adverse health effects of UV radiation are premature skin aging, immune suppression, increased risks of skin cancer, and eye disorders and diseases 2+ ion exists, rather than ‘naked’ Fe2+ ion. This part of the investigation was performed in water as an ionization-supporting solvent, which enables the existence of involved ionic species.Therefore, compounds able to chelate catalytic iron ions are expected to be effective in suppressing the Ka1 = 4.56) than its phenolic OH group (pKa2 = 8.65) 2+:G([FAFe(H2O)5]+), G(H2O), G(FA−), and G([Fe(H2O)6]2+) are the Gibbs free energy of complex, water, ferulate anion, and hydrated Fe2+ ion, respectively. The capacity of FA and 5OHFA to bound Fe2+ ion and monoanionic and dianionic forms of FA and 5OHFA in stoichiometric ratios of 1:1 and 1:2. The preferred coordination site for the Fe2+ ion are carboxylate oxygens of ferulate anion rather than the guaiacyl or catecholic oxygens, in line with the results of Truong and co-workers [rG values also indicate that 5OHFA demonstrates a slightly stronger chelation ability with the Fe2+ ion compared to FA in complexes with a 1:2 metal to ligand stoichiometric ratio. Thus, both FA and 5OHFA possess preventive antioxidant potential via the Fe2+ ion chelation, in such a way to be able to suppress its involvement in the Fenton and Haber-Weiss reactions.-workers . All sturG values by using M06 functional are less exergonic.Presented results for FA complexes are comparable with those recently published by Truong and co-workers . The com•OH radicals with rate constants within the diffusion-controlled limit. Involved mechanisms between two free radicals , which occur on the two PES via MECP, were specifically investigated. A reaction between FAPR and •OH occurs via the RRC mechanism, while 5OHFAPR inactivates •OH via the PCET mechanism. Keto-enol tautomerization, resulting in 5OHFA, proceeds via the hydrogen bond lattice of water molecules, leading to a high increase of reaction rate constant. Results of molecular docking simulations reveal that FA and 5OHFA form a stable complex in the active site of the LOX enzyme. Exergonicity of complexation reactions indicate FA and 5OHFA as potent chelators of ferrous ion.Little is known about the underlying mechanisms of skin protection by FA formulations from UVA-mediated free radicals damage. Besides mechanisms involving enzymes to suppress the formation of free radicals, the sequestration of transition metal ions that catalyse free radical production and direct scavenging could also be operative mechanisms if high concentrations of FA exist at the place of free radical formation. Performed kinetic analysis indicates that FA, 5OHFA, and their phenoxyl radicals effectively scavenge •OH radical formation by sequestering the catalytic Fe2+ ion, and by inhibiting the prooxidative LOX enzyme . Investigated compounds may show a synergistic effect in the antioxidant and inflammatory processes. The possible contribution of those mechanisms to skin protection in vivo is primarily determined by an achievable concentration of antioxidants in situ and in systemic circulation, which must be competitive with other endogenous scavengers and chelators. Inhibition of the prooxidant LOX enzyme, which require much lower concentrations of antioxidants, could be assumed as a more contributing mechanism. Further basic and clinical investigations are needed to determine the exact mechanism(s) underlying the skin protection efficacy of FA.Our results show that FA and 5OHFA have potential to act as multipotent antioxidants via direct free radical scavenging, suppressing


Background: Sudden sensorineural hearing loss is most commonly defined as a sensorineural hearing loss of 30dB or greater over at least three contiguous audiometric frequencies occurring within a 72-hr period. The Astra Zeneca COVID-19 vaccine is suspicious of causing thrombotic complications following its administration, and could theoretically induce hearing loss by damaging the hearing organs through this mechanism, as well as vestibular damage through similar mechanisms.Material and Methods: We reviewed the files of patients with otological symptoms after exposure to the Astra Zeneca COVID-19 vaccine during the year 2021.Case Series: We studied a total of six cases with otologic symptoms temporally related to the Astra Zeneca COVID-19 vaccine. We report four cases of patients presenting with hearing loss and tinnitus a few days after the second dose of the Astra Zeneca vaccine, and one case with the same symptoms after the first dose. Four cases were successfully treated with steroids; however, one case presented to the office two months after the onset of symptoms and did not improve with treatment. We also report the first case of vestibular neuritis temporally related to the administration of the first dose of the vaccine, which also had a good outcome after medical treatment.Conclusions: Prompt treatment in the present cases was a factor associated with a good prognosis. Sudden sensorineural hearing loss (SSHL) is a disease characterized by sensorineural hearing loss (SHL) of unknown cause, most commonly defined as an SHL of 30dB or greater over at least three contiguous audiometric frequencies occurring within a 72-hr period ,2. AlthoSeveral previous reports have mentioned the possible link between COVID-19 infection and inner ear damage ,6-10; hoStudy design: This is a retrospective transversal observational study described as case series.Data collection: The clinical files of patients presenting to our practice in 2021 (the year that the COVID-19 vaccination program started in our country) with auditory and/or vestibular symptoms after the administration of the AZCV were searched in our database and reviewed, obtaining a total of six files for revision. Data analysis: Patients were grouped according to their main otologic symptoms as auditory or vestibular, yielding a total of five patients with auditory symptoms and one patient with vestibular symptoms. No further statistical tests were possible given the small number of cases that we had. We describe cases in detail one by one.Case seriesThe first case is a 61-year-old male patient who presented to the Otolaryngology Clinic in July 2021, referring SSHN associated with severe high pitched tinnitus in the left ear for one month before that initial visit; he had the second dose of the AZCV 10 days before the onset of the otologic symptoms. Pure tone audiometry (PTA) performed 10 days after the onset of symptoms revealed a moderate SHL in the left ear in high frequencies , consistent with the traditional SSNHL criteria , right lateropulsion, and ocular tilt reaction, therefore, presenting the classic clinical signs and symptoms described in VN  and was This is the first case series reporting damage to the hearing organs probably related to a vaccine for COVID-19. Although more evidence is yet to be found, the temporal relation between the AZCV exposure and the appearance of hearing loss and tinnitus a few days after the vaccination in all cases clearly points toward a likely association between the vaccine and the otologic symptoms. Many authors have considered a possible link between COVID-19 infection and SSHL ,6-10; hoWe present six cases of patients with otologic symptoms probably related to the AZCV, all of them with the onset of symptoms a few days after the AZCV exposure; five confirmed to have hearing loss by PTA, and one with clinical criteria strongly consistent with acute VN.A high index of suspicion must be kept in mind for inner ear damage in patients with otologic symptoms after the administration of the AZCV; we recommend these group of patients get a PTA as soon as possible in case of audiological symptoms and start steroid therapy immediately to prevent further damage to the inner ear even if the strict criteria for SSHL are not fully met, in the case of VN it seems reasonable to start treatment as with any other acute VN, at least until further studies are available.Hearing loss and tinnitus can be solved in this context by using appropriate steroid therapy as would be done with any other SSHL. Delaying the treatment with steroids seems to be a bad prognostic factor for recovery in this group of patients with auditory symptoms. VN in this context also seems to have a relatively good prognosis. Pathophysiology of inner ear damage associated with AZCV is yet to be established, further studies are needed to confirm the association of hearing loss, tinnitus, and VN with the AZCV, and to define the optimal treatment protocols in these cases.Damage to the inner ear must be considered as a possible side effect of the AZCV, a high index of suspicion must be observed in this context, and treatment should be started accordingly as soon as possible.


Meanwhile, the energy transfer from PM6 to ITIC-M exists in the active layers; small IE suppresses exciton dissociation. Deteriorating performance of solar cells demonstrates that, except for complementary absorption spectrum and suitable energy levels in PM6:Y6:ITIC-M system, the synergetic effects of carrier dynamics among different organic materials play an important role in influencing the performance of ternary solar cells.Although reported ternary polymer solar cells have higher power conversion efficiency than binary polymers, the mechanism of exciton separation and charge transport in this complex ternary system is still unclear. Herein, based on PM6:Y6:ITIC-M ternary solar cells, we combine the technique of luminescence spectroscopy, including electroluminescence (EL) and photoluminescence (PL) with photovoltaic measurements, to understand clearly the detailed roles of ITIC-M as the third component in the contribution of device performance. The results show that ITIC-M can form the alloy-like composite with Y6 but leave individual Y6 acceptor to conduct charge transfer with PM6 donor, which improves  The development of sustainable energy can help alleviate the energy crisis and environmental problems. In particular, organic solar cells (OSCs), as a kind of strong candidate for the new energy sources, have attracted much attention due to their low cost, light weight, mechanical flexibility, translucency, fast roll-to-roll printing method and non-toxicity ,2,3,4. SocV). However, lower short circuit current (scJ) and fill factor (FF) occur in ternary solar cells, which is contrary to the improved absorption. To account for the mechanism of carrier dynamics in ternary solar cells, EL spectroscopy as a simple measurement was utilized to analyze the carrier recombination by combining with PL spectroscopy. It is demonstrated that electrons will accumulate in the alloy acceptor because of the synergy between inefficient extraction and decreased exciton dissociation in ternary solar cells. Consequently, the increasing recombination leads to a decrease of scJ and FF.In this article, we fabricated various ternary cell devices by incorporating ITIC-M -1-(6-(dicyanomethylene)-2-methyl-5,6-dihydro-6H-cyclopenta[b]thiophen-6-one-5-yl)ethylene)-5,5,11,11-tetrakis(4-hexylphenyl)dithieno -sindaceno dithiophene), an NFA as the additional acceptor material into thiophen-2-yl)-benzodithiophene))-alt-benzodithiophene-4,8-dione)]) PM6:Y6 --3,9-diundecyl-12,13-dihydro-thiadiazolothienothienopyrrolothienothienoindole-2,10-diyl)bis(methanylylidene))bis)dimalononitrile) host blend films. The ITIC-M shows an absorption spectrum from 500 nm to 800 nm and forms the complementary absorption spectrum with PM6 and Y6, finally increasing the photon harvesting of solar cells. Besides, the highest occupied molecular orbital (HOMO) energy level and the lowest unoccupied molecular orbital (LUMO) energy level of ITIC-M are both located between that of PM6 and Y6, respectively, and Y6 and ITIC-M form an alloy acceptor to improve the open circuit voltage  and 1, 8-diiodooctane (DIO) were commercially procured from Sigma-Aldrich, USA, Shanghai branch with purity greater than 99.9% and 98.0%, respectively.−1 PM6:Y6 solution with a weight ratio of 1:1.2 (w/w) and ternary solutions mixed as blend were prepared in CF and then heated and stirred at 45 °C for 3 h. Next, 0.5 v% DIO as additive was added into dissolved solutions an hour before spin coating the active layer. PDINO was mixed into methyl alcohol with concentration of 1 mg mL−1. Ternary solar cells were fabricated in two batches by using the conventional structure of ITO/PEDOT: PSS/active layer film/PDINO/Ag, as shown in Solution preparation and device fabrication: According to the previous report , a 16 mgAs the solutions for ternary devices, in one batch, the polymer donor to the acceptor ratio was kept constant as 1:1.2 and the ratio of Y6 to ITIC-M was changed. In another batch, the ratio of PM6 to Y6 was kept as 1:1.2, and the content of ITIC-M in the bulk-heterojunction (BHJ) was changed. The prepared active layer films and corresponding devices are as follows:Film 1: PM6: Acceptor (Y6:ITIC-M = 1:0) = 1:1.2Film 2: PM6: Acceptor (Y6:ITIC-M = 0.9:0.1) = 1:1.2Film 3: PM6: Acceptor (Y6:ITIC-M = 0.8:0.2) = 1:1.2Film 4: PM6: Acceptor (Y6:ITIC-M = 0.6:0.4) = 1:1.2Film 5: PM6: Acceptor (Y6:ITIC-M = 0:1) = 1:1.2Film 6: PM6:Y6:ITIC-M = 1:1.2:0.1Film 7: PM6:Y6:ITIC-M = 1:1.2:0.2Film 8: PM6:Y6:ITIC-M = 1:1.2:0.4Film 9: PM6:Y6:ITIC-M = 1:1.2:0.6Film 10: PM6:Y6:ITIC-M = 1:1.2: 1Device 1–10: ITO/PEDOT: PSS/film1–10/PDINO/Ag2-filling glove box and the prepared activity was annealed at 80 °C for 10 min and thoroughly dried in vacuum chamber for 1 h. After that, PDINO electron transport layer was spin coated on the active layer at 3000 rpm for 50 s. Finally, Ag cathodes (with the film thickness of 1000 Å) were deposited on the top at rate of 1.5 Å/s under a vacuum pressure of 2 × 10−4 Pa. The active area of solar cells is 4 mm2.First, indium tin oxide (ITO) coated glass substrates (sheet resistance of 15 ohm/square) were cleaned ultrasonically in a cleaning agent, deionized water and ethanol for 30 min, respectively, and then immediately blow-dried by high-density nitrogen, followed by plasma UV processing for 90 s. A highly conducting polymer PEDOT:PSS was spin coated onto the treated ITO substrates from an aqueous solution at 5000 rpm for 30 s, then these ITO coated with PEDOT:PSS were annealed at 150 °C for 10 min. Next, the solution used as active layer was prepared through spin coating at 3000 rpm for 30 s in NJ-V characteristic curves of solar cells were measured with a Keithley 2400 source equipment unit under AM 1.5 G simulated solar illumination with an intensity of 100 mW cm−2. The EQE measurements were performed using a QE/IPCE Measurements Solar Cell Scan 100  system equipped with calibrated silicon photodiode as the reference cell. PL spectra were acquired using a modified Horiba FL1000. EL spectra were performed by using Keithley 2410 to support bias and detectors in Horiba FL 1000. The UV−vis absorption spectrum was acquired on Shimadzu UV-3101 PC spectrometer. The surface morphology characteristics and 3D images were measured by atomic force measurement .Measurement and characterization: The The device structure and HOMO energy level and LUMO energy level of component materials used in devices are shown in The absorption spectra of pure films and D:A blend films were measured and the normalized absorption spectra are shown in J-V) characteristics of different devices under AM1.5G illumination with the intensity of 100 mW cm−2. scJ of 24.80 mA cm−2, ocV of 0.83 V and FF of 68.30%. ocV increases with the augment ratio of ITIC-M in acceptors from 0.83 V to 0.91 V (device 1 to device 4), and obtains the highest value of 1.02 V in PM6:ITIC-M binary solar cell (device 5). The same ocV increase (from 0.85 V to 0.91 V) can be observed in devices 6 to device 10, which is attributable to the alloy acceptor formed by Y6 and ITIC-M as shown in later discussion. However, scJ and FF decrease sharply with addition of ITIC-M and finally the value of PCE decreases. We determined the shunt (Rsh) and series (Rs) resistances of these devices from their J-V curves (s), as well as the leakage current and free carrier recombination in their BHJ (Rsh) of the devices. The values of Rs in devices incorporating ITIC-M are higher than PM6:Y6 binary device (device 1), implying more defects at the interfaces and within the BHJ blend films of these devices. Moreover, lower values of Rsh suggest higher degrees of free carrier recombination. V curves  to evaluAs external quantum efficiency (EQE) results show in −1 in the out-of-plane direction, which corresponds to that of PM6. The almost identical locating at the same position and same intensity of GIXRD peak indicates that ITIC-M generates nearly no difference in polymer (PM6) crystallinity.The microscopic morphology of the active layer plays a decisive role in the generation and transmission of charges in devices ,25. In oCT) vary in different devices; the ECT [CT also changes from 1.46 eV to 1.50 eV with the increase of ITIC-M content. The changes of ECT indicate that it is plausible to consider Y6 and ITIC-M forming an alloy acceptor, and the ECT reaches maximum (1.67 eV) in PM6:ITIC-M binary device, which is consistent with the variation of ocV.According to the above results, the carrier dynamic inside devices has changed after the addition of ITIC-M and the new carrier dynamic leads to a decreasing performance. To further understand the charge separation and charge transport process in ternary devices, we used EL spectrometry to study the exciton decay way and the charge transfer states (CTSs) of devices. The EL spectra of devices with different content of ITIC-M are shown in  the ECT ,27 becomscJ and FF. Therefore, it is concluded that by adding ITIC-M, even the exciton of PM6 is dissociated easily, but the recombination probability of CT states of PM6:Y6:ITIC-M increased, which may be due to the deterioration of charge transportation and increase of defect density.PL spectra were measured to further analyze the energy transfer mechanism in PM6:Y6:ITIC-M blend film excited by 550 nm as shown in CT emission of PM6:ITIC-M almost disappears. According to the result of multiple peaks Gaussian fitting , ε0 is the vacuum permittivity and tn is the trapping state density.The hole mobility and the electron mobility of ternary devices incorporated with ITIC-M all decrease dramatically. In addition, the unbalance between hole and electron mobility would lead to the accumulation of carriers with the lower mobility in the device. This will result in an additional electric field which would hinder the extraction of carriers and increase the radiative recombination ,34. ThenV curves . When th17 cm−3, 1.08 × 1017 cm−3 and 4.84 × 1016 cm−3 and 2.36 × 1016 cm−3, 4.13 × 1016 cm−3 and 2.07 × 1016 cm−3, respectively. The variation of hole trapping states is coherent with that of hole current that changes a little with the increasing of ITIC-M content. However, even the electron trapping state density decreases with the increasing of ITIC-M content in the active layer, but the electron current decreases dramatically. This means that the electron transport is going to be difficult in the active layer due to the adding of ITIC-M, which is proven by the lower electron mobility in ternary devices with increasing of ITIC-M. It also corresponds to the electroluminescence results detected in PM6:Y6:ITIC-M active layer.The electron and hole trapping states of devices 1, 4 and 8 are acquired as 1.77 × 10phJ) versus the effective voltage (effV) curves for the devices 1, 4 and 8, plotted in phJ is calculated according to [LJ and DJ represent current density under AM 1.5G illumination and in the dark, respectively. effV is calculated according to [V0 stands for the voltage at which phJ = 0 and aV is the applied voltage.Otherwise, we measured the photocurrent density Jprding to ,37:(4)VephJ of three devices shows linear dependence on the voltage at a low effV (<0.1 V), and rapidly reaches saturation at the high effV (>1 V). It clearly shows that the devices 4 and 8 have a lower saturation photocurrent density (satJ) than that of device 1. The charge dissociation probability P  of both devices determined by phJ/satJ under short circuit condition is 89.11%, 84.27% and 84.69%, respectively. Combining P  and dark J-V curves  coexistence of EL peaks at 1.46 eV and 1.53 eV means that parts of ITIC-M and Y6 formed alloy acceptor while individual Y6 exists and conducts charge transfer with PM6 alone, (ii) large overlap of PM6 PL (678 nm) and ITIC-M absorption (700 nm) indicates existence of energy transfer from PM6 to ITIC-M. Small IE is adverse to exciton dissociation and unbalance of carrier mobility leads to accumulation of electrons, finally improving recombination. Except complementary absorption spectrum and suitable energy levels in PM6:Y6:ITIC-M system, carrier dynamics between organic materials play an important role in the performance of ternary solar cells.