Mycoplasma hyopneumoniae (M. hyopneumoniae) is the primary agent of enzootic pneumonia in pigs. Pigs are often infected with different M. hyopneumoniae strains. This study assessed the efficacy of vaccination against experimental infection with two genetically different M. hyopneumoniae strains in weaned piglets. At 33 days of age (D0), 45 M. hyopneumoniae-free piglets were randomly assigned to three different groups: 1) negative control group : not vaccinated, not infected, 2) positive control group : not vaccinated, infected, and 3) vaccination group : single vaccination with an inactivated whole-cell M. hyopneumoniae vaccine  (D1), infected. The PCG and VG were endotracheally inoculated with 7 × 107 CCU in 7 ml of the highly virulent M. hyopneumoniae strain F7.2C (D24) and 7 × 107 CCU in 7 ml low virulent strain F1.12A (D25). A respiratory disease score (RDS) was assessed from D24 until D53. At D53 (euthanasia), macroscopic lung lesions (MLL) were scored, log copies of M. hyopneumoniae DNA (qPCR) and IL-1 and IL-6-concentrations (ELISA) on bronchoalveolar lavage fluid were determined.P < 0.001) and 0, 7.56 and 0.68 (P < 0.001) for NCG, PCG and VG, respectively. The qPCR results for PCG and VG were 3.99 and 1.78 log copies (P < 0.001), respectively, with a significant difference between PCG and VG. The IL-1 and IL-6 results at euthanasia for NCG, PCG and VG were 17.61, 1283.39 and 53.04 pg/ml (P < 0.001) and 148.10, 493.35 and 259.80 pg/ml (P = 0.004), respectively with a significant difference between PCG and VG.The RDS and MLL at euthanasia were respectively 0, 1.20 and 0.55 (M. hyopneumoniae strain as the vaccinated pigs coughed significantly less, and showed significantly less lung lesions compared to the non-vaccinated challenged pigs: the vaccinated animals showed a 52.9% lower RDS and 91.0% lower MLL compared to the PCG. In the bronchoalveolar lavage fluid collected at the necropsy of the vaccinated pigs, a significantly lower amount of M. hyopneumoniae-DNA and a significantly lower IL-1 and IL-6 concentration was found compared to the pigs of the PCG.Vaccination with Hyogen® in pigs was efficacious against an experimental challenge with both a low and highly virulent  Mycoplasma hyopneumoniae (M. hyopneumoniae) is the causative agent of enzootic pneumonia . d, USA)) . This paM. hyopneumoniae organisms [M. hyopneumoniae DNA of strain F7.2C was used to convert the threshold values to the number of M. hyopneumoniae organisms. Values below the dilution of 1.50 × 101 (1.18 log copies) were considered as negative.Bronchoalveolar lavage fluid was collected from all animals two weeks PI (D39) by inserting a catheter  in the trachea by means of a mouth gag and whilst snaring the pigs. Next, the lungs were flushed with 20 mL sterile PBS to collect the bronchoalveolar lavage fluid. Additionally, during necropsy (D53), bronchoalveolar fluid was collected from the left part of the lung before collection of the histopathological samples by flushing the head bronchus of the left part of the lung with 20 mL sterile PBS. The bronchoalveolar lavage fluid was divided into seven aliquots and stored at −70 °C awaiting analysis. From one aliquot of the bronchoalveolar lavage samples, DNA was extracted with the QIAGEN protocol  and quantitative PCR (qPCR) was performed as previously described to detect the numbers of rganisms . A tenfoM. hyopneumoniae-specific antibodies in bronchoalveolar lavage fluid collected at D39 and D53 was determined via an indirect ELISA  according to the protocol of Bereiter et al. [M. hyopneumoniae antigens on which the bronchoalveolar lavage fluid  was added, undiluted. Peroxidase labeled goat anti-porcine Ig A or Ig G polyclonal antibodies  were added and the OD at 450 nm was measured.The isotype of r et al. , 24. BriThe bronchoalveolar lavage fluid collected at D39 and D53 was tested undiluted for presence of porcine TNF-α , IL-1  and IL-6 . A sandwich ELISA was performed according to the manufacturers’ recommendations. The sample reactions were measured using OD at 450 nm and quantified by the use of a standard curve as described in the manual.M. hyopneumoniae with a blocking ELISA , according to the protocol manual. Sera with optical density < 50% of the average value of the OD-buffercontrol were considered to be positive. All values above or equal to 50% of the average value of the OD-buffercontrol were classified as negative.At D1, D24 and at euthanasia (D53), blood was collected from all pigs and tested for the presence of antibodies against Staphylococcus pseudintermedius streak [. Plates were incubated overnight in a 5% CO2-enriched environment at 35 °C for 48 h for identification of respiratory bacteria in the lungs.For the bacteriological examination, ten μL of bronchoalveolar lavage fluid collected on D53 of each pig was inoculated on Columbia agar supplemented with 5% sheep blood  with a s streak . Plates M. hyopneumoniae, TNF-α, IL-1, IL-6, Ig A, Ig G and qPCR results were analyzed using a non-parametric Kruskal-Wallis, as the data did not fulfil the assumptions of normality. Theses analyses were performed with SPSS 22 for Windows . Percentage of seropositive pigs and percentage of pigs testing positive with qPCR in each group were analyzed using binomial logistic regression (R version 3.3.1) [P < 0.05.Descriptive statistics were performed in order to check the normality of the data. One-way analysis of variance (ANOVA) was used to analyse weight and ADG. Repeated measurements ANOVA was performed to analyze the RDS data. Scheffé’s post hoc test was used to make pairwise group comparisons. The parameters MLL, histopathological lesions, percentage air analysis, ELISA n 3.3.1) . ResultsHaemophilus parasuis was isolated in pure culture from the meninges and pericardium. All animals in the PCG coughed, showed macroscopic lung lesions and seroconverted. Mycoplasma hyopneumoniae-DNA was detected in 19 out of 20 animals from this group.No animals died during or shortly after the inoculations. One out of 45 piglets in the NCG was euthanized (D22)  due to nervous symptoms and lateral decubitus. Necropsy was performed and 24–53 was 1.20 ± 0.83 and 0.55 ± 0.42 for the PCG and VG (P < 0.001), respectively. This corresponds with a reduction of 52.9% in RDS when vaccinating the piglets compared to the PCG. All groups significantly differed from each other. These results and the RDS 1–53 and RDS 1–23 results are shown in Table The results of the clinical parameters are summarized in Table The average group weight at D0, D24 and D53 and the ADG calculated during these three periods  for each group are shown in Table P < 0.001) (Table P < 0.001), respectively. The percentage of lung area occupied by air (percentage air) was 47.72 ± 7.29, 34.49 ± 8.22 and 45.23 ± 5.99% for the NCG, PCG and VG (P < 0.001), respectively. For the MLL and percentage of air analysis, the PCG was significantly different from the VG and NCG. For the histopathology score, all groups significantly differed from each other  and 1.78 ± 1.36 (VG) (P < 0.001) at D53 . At D53, the results for Ig A were 0.18 ± 0.14, 2.10 ± 0.75, 2.08 ± 0.58 for the NCG, PCG and VG, respectively (P = 0.005). Both the Ig G and Ig A concentrations at D53 of the PCG and VG differed significantly from the NCG.At D53, the Ig G values were 0.38 ± 0.17, 2.25 ± 0.91 and 2.41 ± 0.88 for the NCG, PCG and VG, respectively .At D53, the IL-1 concentration in the bronchoalveolar lavage fluid was 17.61 ± 1.98, 1283.39 ± 1075.65 and 53.04 ± 54.23 pg/ml in the NCG, PCG and VG, respectively (P = 0.004) with significantly higher concentrations in the PCG compared to the NCG and VG.At D39, the IL-6 concentration was significantly different between the PCG and VG Table . At D53,M. hyopneumoniae. The OD values at D53 were 0.90 ± 0.10, 0.23 ± 0.11 and 0.087 ± 0.041 in the NCG, PCG and VG, respectively (P < 0.001). In the VG significantly lower OD-values were detected, thus higher M. hyopneumoniae specific antibodies compared to the PCG and NCG  pneumonia lesions and fissures [The challenge infection was performed with two genetically different  M. hyopna et al.  and botha et al. , Vranckxa et al.  and Staka et al. , respectnfection . Taken tnfection , 31–38 anfection , 31–35. fissures . The curThe pigs in the vaccinated group coughed significantly less (−53%) from inoculation until euthanasia compared to the positive control group, demonstrating the efficacy of the vaccine. The degree of coughing is an important efficacy parameter , 40 not The MLL score in the vaccinated pigs was very low 0.68) and statistically different from the PCG (7.56), implying a reduction with 91%, and demonstrating the efficacy of the vaccine. A recent field study showed that when pigs are infected with more strains, more lung lesions are detected 8 and sta), higherM. hyopneumoniae results in intrabronchiolar- and bronchial exudate and infiltration of lymphohistiocytic cells in the lung tissue. These responses may result in compressing the airways and alveoli, leading to less air volume in the lungs [M. hyopneumoniae DNA in the bronchoalveolar lavage fluid compared to the non-vaccinated animals (1.78 versus 3.99), which may lead to less shedding of M. hyopneumoniae in vaccinated pigs. Similar reductions were found in previous studies [In the vaccinated animals significantly less lymphohistiocytic infiltration (1.92) was observed compared to the PCG 3.32). These results are in accordance with the results of previous studies , 31, 32.. These rhe lungs . Vaccina studies , 44. TheMycoplasma hyopneumoniae-specific antibody (Ig G and Ig A) levels in tracheo-bronchial washings were not significantly different between vaccinated and non-vaccinated pigs. Our findings are in accordance with Djordevic et al. (1997) [M. hyopneumoniae- antibodies induced by vaccination into the bronchoalveolar washings might be implied in resolving mycoplasmal pneumonia [M. hyopneumoniae-specific antibodies in trachea-bronchial washings.. (1997) , 45, butneumonia , 46, 47.M. hyopneumoniae and the exact mechanisms in which way M. hyopneumoniae-bacterins provide protection to the animal.There were no significant differences between the vaccinated and non-vaccinated animals for the TNF-α concentration in the bronchoalveolar fluids. In the study of Marchioro et al. , vaccinaM. hyopneumoniae infection must be kept into account causing the challenge infection to be more successful. The IL-1 and IL-6 concentrations at necropsy in the vaccinated pigs were significantly lower than in the non-vaccinated pigs and not significantly different from the levels in negative control group. These results substantiate the statement that was made in Marchioro et al. [M. hyopneumoniae vaccination [The cytokines IL-1 and IL-6, next to TNF-α are pro-inflammatory cytokines . Previouo et al.  based oncination .M. hyopneumoniae outbreaks [M. hyopneumoniae infection, such as PRRSv Pasteurella multocida, and Actinobacillus pleuropneumoniae. [M. hyopneumoniae vaccination as well [M. hyopneumoniae and no vaccinations against M. hyopneumoniae in the sows, nor the piglets were performed. This is a double sided given. On the one hand, Hodgins et al. [M. hyopneumoniae in the face of antigen-specific maternal-derived immunity results in cell-mediated immunity priming and anamnestic cell-mediated immunity responses following the exposure to M. hyopneumoniae antigen. On the other hand, some factors in the experimental study were comparable to field circumstances or challenged the vaccine more. First, although the piglets were free from certain pathogens, other than that, the piglets were originating from a conventional farm. Second: the piglets were transported from the herd of origin to the experimental facilities and the piglets needed to establish a new hierarchy after comingling. These events are associated with stress, which might influence the efficacy of the vaccine. This can resemble the stress that piglets experience in the field, when sorted and moved from one facility to another in a multi-site production system. Thirdly: the vaccine was highly challenged in this experimental study, because as stated by Villarreal et al., [M. hyopneumoniae challenge dose used to infect the pigs was higher than might be reached under natural conditions resulting in a faster and higher colonization level and presumably challenging the vaccine more than under field circumstances [M. hyopneumoniae infected pigs should be done with caution, this infection model enabled to study the effect of the vaccine in M. hyopneumoniae infected pigs in a reproducible and standardized way.In this study, the pigs were vaccinated approximately 4 weeks before challenge, as vaccination is most likely to be effective if active immunity is established before the piglets are exposed to the pathogen. Under field conditions, however, the time between vaccination and infection is not known, and very likely highly variable between pigs. Hence, it is possible that the vaccine used in the field is less efficacious when the timing of vaccination is not optimal. As this is an experimental study, the results cannot be extrapolated as such to field conditions. Under field circumstances, more factors challenging the efficacy of a vaccine are present, compared to the controlled environment of an experimental facility. First, multi-factorial and multi-pathogen enzootic pneumonia outbreaks are typically found in the field, rather than isolated utbreaks . The pigumoniae. , 53, 54  as well . Second, as well , it couls et al.  stated ts et al.  showed t et al.,  and Meyn et al., , it can mstances , 37. AltM. hyopneumoniae strain, one-dose vaccination of piglets was efficacious for the most part, as coughing was reduced by more than 50% and macroscopical lung lesions by 91%. In addition, the lymphohistiocytic infiltration in lung tissue was lower, the number of log copies of M. hyopneumoniae DNA detected in bronchoalveolar lavage fluid and the IL-1 and IL-6 concentrations at euthanasia were lower in the vaccinated animals compared to non-vaccinated animals.Using a double challenge infection model with a low and highly virulent


Mortierella alpina aging process remains elusive. Herein, subcellular proteomics was employed to unveil the composition and dynamics of the LD proteome in the aging M. alpina for the first time. More than 400 proteins were detected in LDs and 62 of them changed expression significantly during aging. By combining the LD proteomic data with whole-cell data, we found that the carbohydrate metabolism and de novo lipid biosynthesis were all inhibited during aging of M. alpina mycelia. The up-regulation of fructose metabolism-related enzymes in LDs might imply that LDs facilitated the fructose metabolism, which in turn might cause pyruvate to accumulate and enter malate-pyruvate cycle, and ultimately, provide additional NADPH for the synthesis of arachidonic acid (ARA). Lysophospholipase and lecithinase were up-regulated in LDs during the aging process, suggesting that the phospholipids and lecithin were starting to be hydrolyzed, in order to release fatty acids for the cells. The impairment of the anti-oxidant system might lead to the accumulation of ROS and consequently cause the up-regulation of autophagy-related proteins in LDs, which further induces the M. alpina mycelia to activate the autophagy process.Lipid droplets (LDs) participate in many cellular processes in oleaginous microorganisms. However, the exact function of LDs in the  Lipid droplets (LDs), are intracellular organelles characterized by large deposits of neutral lipids, and consist of a neutral lipid core surrounded by a phospholipid monolayer1245657825Mortierella alpina, a filamentous fungus, is viewed as a particularly good producer of ARA-rich oilM. alpina, which employ an additional culture step after regular fermentation, which is conducted in an environment without carbon source, has long been known as an effective way to improve ARA contentArachidonic acid  is an important polyunsaturated fatty acid which shows various physiological functions in the human body and has broad applications in medicine, cosmetics, and other fields1112M. alpina LD proteins and their associations with the whole-cell signal pathways may be a key step to understand the exact mechanism of ARA accumulation in the aging M. alpina mycelia. Therefore, we used a subcellular proteomics approach to characterize the composition and changes of the LD proteome during the M. alpina aging process. Moreover, we further combined whole-cell proteome data published separately, with the LD proteome data from this study to identify the associations between the whole-cell signal pathways and the dynamics of the LD proteome in aging M. alpina cells. Our findings show that LD-associated proteins have diverse functions involved in lipid metabolism and the dynamics of LD proteome are influenced by some crucial signal pathways. These results will pave the way for a detailed understanding of the molecular mechanisms of M. alpina aging and help to improve the aging technology for ARA production.Since LD-associated proteins are involved in various cellular processes, such as lipid metabolism and LD size control4M. alpina mycelia entered the aging period after around 156 h of fermentation, when glucose was exhausted , most of the pearance . After spearance . Lipid dpearance . At 192 pearance . TEM obspearance . TarnopoAccording to Ding and coworkers’ method17M. alpina mycelia at this time point. Moreover, in our other work, we found that at the end of the aging process (240 h) the mycelia began to decompose and cytoplasm seemed to have leaked outSince the percentage and concentration of ARA increased remarkably at the middle stage of the aging process (192 h) , signifiM. alpina LD proteome were actin, elongation factor 1-alpha, ATP synthase subunit beta, Histone H4, Histone H2B, Tubulin alpha-1C chain and proteins related to the elongation of fatty acids , 30 proteins showed significant differences in abundance (13 up-regulated and 17 down-regulated) in the aging group as compared to the control group  were up-regulated in the LDs during the aging process . Since aM. alpina mycelia could accumulate sucrose prior to entering the aging process and the accumulated sucrose was subsequently catabolized quickly at the early stage of agingM. alpinaM. alpina.In our previous work, we found that de novo fatty acid biosynthesis, were found to be down-regulated both in the LD and the whole-cell proteomes, suggesting that de novo fatty acid biosynthesis was significantly weakened in M. alpina during the aging period. This was also confirmed by the results of lipid analysis  was up-regulated in the LD proteome of the aging cells, while it remained undetectable in the whole-cell proteome . Lysopho31As shown in M. alpina mycelia remarkably increased during the aging processM. alpina mycelia towards autophagy. We previously found that some of the M. alpina mycelia appeared to decompose at the end of the aging processM. alpina mycelia were undergoing active, and perhaps even runaway autophagy. Overall, the expression of autophagy-related proteins in LDs suggests that these organelles may themselves participate in the autophagy mechanism of M. alpina, and thus possess a nutrient recycling role necessary to prolong the survival of mycelia undergoing carbon starvationIn addition, some differentially expressed enzymes related to other metabolic pathways  were also found in both the LD and the whole-cell proteome datasets. Notably, most of the enzymes involved in glutathione metabolism and oxidative phosphorylation were significantly down-regulated. Glutathione plays a key role in preventing damage caused by reactive oxygen species (ROS) in fungiM. alpina mycelia significantly changed during the aging process. Using the subcellular proteomics method, the composition and dynamics of the LD proteome in aging M. alpina cells was investigated. M. alpina LDs contain a large number of different enzymes which participate in diverse metabolic pathways. By combining the LD proteomic data with whole-cell proteomic data, we found that enzymes related to carbohydrate metabolism and de novo lipid biosynthesis were all suppressed. The upregulation of enzymes related to fructose metabolism implies that LDs facilitate fructose utilization, which might cause pyruvate to accumulate, enter the malate-pyruvate cycle, and ultimately, provide additional NADPH for the biosynthesis of ARA. The increased expression of lysophospholipase and lecithinase suggests that the LDs started to hydrolyze their phospholipids and lecithin, releasing the resulting fatty acids for mitochondrial energy metabolism. At the same time, the impairment of antioxidant systems might lead to the accumulation of ROS, which in turn can induce the expression of autophagy-related proteins in the LDs, which might further mediate the activation of autophagy in M. alpina mycelia. We believe these findings will be helpful for deciphering the exact function of LDs during the M. alpina aging process and pave new ways for the efficient production of ARA and other polyunsaturated fatty acids via controlled mycelial aging.In summary, we found that the morphology of LDs in M. alpina R807 (CCTCC M2012118) was purchased from the China Centre for Type Culture Collection. The potato dextrose agar consisted of (g/L): potato extract 200; glucose 25; agar 20. The seed medium contained (g/L): glucose 30; yeast extract 6; NaNO3 3; KH2PO4 3; MgSO4.7H2O 0.5. The fermentation medium contained (g/L): glucose 80, yeast extract 10, KH2PO4 4, NaNO3 3, MgSO4.7H2O 0.6.M. alpina were the same as those reported in our previous workM. alpina mycelia was taken from a seed tube containing 30% glycerol and stored at −80 °C, to transferred onto PDA slants. The cultures were subsequently incubated in an electro-thermal incubator for one week at 25 °C. When the PDA slant surface was covered with white mycelia, the mycelia were harvested for seed culture. 500 mL baffled flasks containing 100 mL fresh medium were used for seed and fermentation culture. Seed culture was conducted for 24 h, after which 10% (v/v) of the culture broth was used to inoculate the fermentation broth. After regular fermentation, mycelia were cultured continuously without carbon source to induce the aging process. All cultivations were conducted at 25 °C, initial pH 6.0, under constant orbital shaking at 125 rpm.The culture and aging conditions for The experiments were conducted according to a reported methodThe samples for transmission electron microscopy (TEM) observation were prepared as follows: after being cut into small pieces, mycelia were prefixed in 2.5% (w/v) glutaraldehyde in PBS (pH 7.4) at 4 °C. In order to remove residual glutaraldehyde, 0.1 M PBS (pH7.4) was used to wash the samples three times for 15 min each. Subsequently, 2% (w/v) osmium tetroxide  was used to post-fix the samples at room temperature for 1 h. Sample dehydration was conducted via subsequent rinses with serially higher ethanol concentrations of 35%, 50%, 70%, 90%, 95% and 100%. After embedding in epoxy resin , samples were prepared as 90 nm sections using an EM UC6 Ultramicrotome . After double staining with uranyl acetate  and lead citrate , the sections were observed using a JEM-1011 TEM .et al. with minor modifications2, pH7.4), after which the samples were centrifuged at 182000 g for 1 h at 4 °C. The floating LDs were collected in a 1 mL Eppendorf tube and retained on ice during the following washing steps. 1 mL of the solution was carefully taken from the middle of the gradient and further centrifuged at 270000 g for 30 min at 4 °C, and the resulting supernatant was collected as the cytosol fraction for determination of purity. The pellet at the bottom of the tube was washed three times with 1 mL of buffer B and resuspended in 1 mL of buffer B to obtain the total membrane fraction. The isolated LDs were suspended in buffer B and stained with Nile Red solution  or left unstained, for observation under an optical microscope  and a confocal microscope .Lipid droplets (LDs) were isolated according to the method reported by Ding 2O  solvent system, respectively. The samples were visualized using iodine vapor.Neutral lipids and polar phospholipids were separated by silica gel thin-layer chromatography using a hexane-diethyl ether-acetic acid  and a chloroform-methanol-acetic acid-HAccording to the reported methodThe protein samples of the LDs were separated by SDS-PAGE, the identified LD protein bands cut out of the gel, destained twice with 200 μL acetonitrile–25 mM ammonium bicarbonate  and washed 3 times with UA buffer  for protein digestion.et al.4HCO3. The resulting protein suspension was combined with 40 μL 25 mM NH4HCO3 containing 3 μg trypsin  and incubated at 37 °C overnight for digestion. The resulting peptide concentration was determined via the optical density at 280 nm.Protein digestion was conducted using the methods described by Wisniewski C18 Cartridges  were used to desalt the peptides in each sample, which were subsequently concentrated by vacuum centrifugation and reconstituted in 40 μL 0.1% trifluoroacetic acid . To conduct the MS experiments on a Q Exactive mass spectrometer , samples comprising 5 μg of total peptides was loaded onto a the C18-reversed phase column  in buffer A  and then separated with a linear gradient of buffer B . To obtain the MS data, a data-dependent top10 method was used to choose the most abundant precursor ions from the dynamic survey scan for HCD fragmentation. The target value was determined based on predictive Automatic Gain Control. For each sample, the MS experiments were conducted three times.® homogenizer , and subsequently boiled for 5 min. The samples were then ultrasonicated and boiled for another 5 min. After centrifugation at 14 000 rpm for 15 min, the resulting supernatants were collected and the corresponding protein concentrations were measured using a BCA Protein Assay Kit  with BSA as standard. The protocols for protein digestion and LC-ESI-MS/MS were same as above.To obtain the whole-cell proteomics data, aliquots of the mycelia harvested by centrifugation for the lipid droplet proteomics experiment were used. The experimental protocol and whole-cell proteomics data are presented in other worke-3 were retrieved for each query sequence and finally loaded into Blast2GO (version 2.7.2) to analyze the related KEGG pathways4243The MaxQuant software suit (version 1.3.0.5) was used to analyze the MS data, which were searched against the UniProtKB database . An initial search was set at a precursor mass window of 6 ppm. This search was conducted with the enzymatic cleavage rule of Trypsin/P with a maximum of two missed cleavage sites allowed. Carbamidomethylation of cysteines was defined as fixed modification, while protein N-terminal acetylation and methionine oxidation were defined as variable modifications. The cutoff of the global false discovery rate (FDR) for peptide and protein identification was set at 0.01. Label-free quantification was conducted in MaxQuant according to the methods described previouslyHow to cite this article: Yu, Y. et al. The Role of Lipid Droplets in Mortierella alpina Aging Revealed by Integrative Subcellular and Whole-Cell Proteome Analysis. Sci. Rep.7, 43896; doi: 10.1038/srep43896 (2017).Publisher's note: Springer Nature remains neutral with regard to jurisdictional claims in published maps and institutional affiliations.


ADL). The main objective of this study was to develop and verify the methods required to predict and investigate the ADL. The variables derived from the wearable sensors were used to create a MNG) obtained from frequency domain analysis. The MNG provides a means to assess aerobic fitness. The predicted ADL was strongly correlated  with the measured −1·kg−1. The MNG calculated based on predicted r = 0.71, P < 0.001) with MNG calculated based on measured Currently, oxygen uptake ( ADL) have the potential to identify not only changes in physical fitness, but also disease states before the manifestation of clinical symptoms5The measurement of oxygen uptake (ML) techniques are becoming popular to analyze the large quantities of longitudinal data streamed from these devicesML algorithms may provide the technical basis to better identify non-trivial and complex patterns in long-term continuous biological signalsML is often based on the relationship between known inputs and outputs (supervised learning)In parallel with the advances in wearable devices, machine learning . A written, informed consent was obtained from all participants. The Office of Research Ethics at the University of Waterloo reviewed and approved the research procedures (ID: ORE20931) that were consistent with the Declaration of Helsinki.ADL. Considering a step duration of 30 s, the PRTS was generated according to previous literature1314−1). These levels corresponded to ≈ ±30% of the normal walking cadenceADL protocol (≈20 min) was composed by sitting, organizing the shelf, carrying objects (≈4.5 Kg), stairs (four up and four down flights of stairs), self-paced walking and sitting using the computer. As opposed to previous studies91011®). The raw sensor signals were used to obtain heart rate (HR), minute ventilation (BF), total hip acceleration (Hacc), and walking cadence (CAD) through previously validated proprietary algorithmsHR data, a new variable was derived. The ΔHR was composed by the difference between the current HR value and the previous value by a 1 s lag operator, capturing dynamic changes in cardiac activity. The combination of the accelerometer sample rate (64 Hz), resolution (0.004 g) and range (16 g’s) was sufficient to capture all expected ADL movementsThroughout the PRTS and simulated ADL, the HR, ΔHRBF, Hacc, CAD and As demonstrated in r) of all folds from the validation process. The time series data and the ability of the predictor to estimate the system temporal dynamics (further explained) were considered into this validation.As described in Code I , the tesEnsemble models  have gained popularity for outperforming singular models with large complex dataHR > 50 bpm). For each time point, the feature values were evaluated by traversing the nodes to the bottom of the tree based on their decision values. Each bottom node, the “leaf node”, contains the tree’s estimated output for the given feature values. Each regression tree was grown individually with a randomly sampled subset of the training data. The final estimated For this study, the random forest model was implemented as an ensemble of bootstrap aggregate regression trees. Specifically, each tree is made up of nodes with up to two children nodes, starting with the root node and traversing down to the end. A node contains a splitting criterion  T individual regression trees. Each individual regression tree was trained on a random data sample (in-bag selection) for generalizability. Each tree was grown node-by-node as follows. For each node, a random 1/3 subset of the features was selected as candidate splitting features. An optimal node split (into left and right subtrees) was sought such that it minimized the sum of squared residuals in the two prospective subsets:Mathematically, let s is the splitting value, xi is the candidate splitting feature, yi, and where This process was repeated recursively for each node, until a full tree was grown. Thus, given a new feature vector x, each tree predicted the Since each random forest, one per participant, was validated individually, a total of sixteen random forest were generated at the end of the leave-one-participant-out cross-validation , Code I.Hacc data were considered as system inputs and the measured and predicted The data corresponding to the PRTS protocol, optimized for system identification13MNG) was used as an index of the system temporal dynamics. Higher MNG values mean faster aerobic responses. The algorithm for MNG calculation is described in the As a characteristic of the PRTS protocol1320242526ADL using the raw measured MNG estimated from the predicted MNG estimated from the measured r coefficient, Bland-Altman plot, confidence interval  estimated from the measured RMR) estimated from the 60 s average of the ADL that is in majority composed by light and moderate intensityMETS higher than 6.0. Therefore, the average of the samples within the intervals 2–3.9 (505 ± 137 samples per participant) and 4.0–5.9 (422 ± 67 samples per participant) were grouped as light and moderate activities, respectivelyFor each participant and considering the entire group response, the predicted ADL are displayed in r = 0.87, P < 0.001 and n = 20,868) with the measured data during ADL. By individually analyzing the correlation level, all participants presented a strong and significant positive correlation  between predicted and measured data with a bias of 0.331 ± 1.187 ml·min−1·kg−1. The Bland-Altman plot for the measured and predicted ADL is shown in ADL, the bias  was statistically (P < 0.05) higher than the equality line. The −1·kg−1 around the bias. The relative distribution of the error is plotted in r = 0.69, P < 0.001 and n = 12,480). By individually analyzing the correlation level, all participants presented a strong and significant positive correlation  between predicted and measured data. The Bland-Altman plot for the measured and predicted −1·Kg−1 was lower (P < 0.001) than the equality line representing only ≈1.7% of the average response during the PRTS protocol. The −1·kg−1. The relative distribution of the error is plotted in Considering all data points from all participants during PRTS , the corRMR) and during light and moderate ADL is depicted in ADLMETS), therefore these data were excluded a priori. The proximity of the estimated METS to the equality line demonstrates that the random forest was able to dissociate between different metabolic demands. The proposed algorithm can be used to classify activity levels between light and moderate ADL.The ability of the random forest algorithm in estimate different levels of metabolic equivalent at rest  and strongly, positively correlated to the MNG calculated from measured MNG calculated from predicted MNG response. The bias was statistically (P = 0.012) lower than the equality line (bias = 0). The MNG response).The comparison of the aerobic system temporal dynamics assessed by the protocol  is displMNG from the predicted oxygen uptake were similar to those of oxygen uptake measured by a portable metabolic device. In addition, the random forest algorithm was able to identify physical activity levels and the resting metabolic demand.In agreement to the initial hypothesis, the signals obtained from the wearable sensors allowed the prediction of oxygen uptake during activities of daily living and random paced walking. Aerobic system temporal dynamics assessed by the ADL represent a challenge for the precise physical activity estimation in a realistic scenario9323334Estimating the correct measurement of physical activity level during realistic scenarios remains a challengeADL protocol as well as an over-ground walking protocol (PRTS) that mimicked the dynamic changes in walking cadences expected during daily activities. The PRTS protocol offered an optimized stimulus for the aerobic system analysis through the study of the During randomly varying exercise intensities, assessment of the rate at which et al.ADL. However, the ability of the algorithm to correctly identify the Recently, Altini HR and accelerometer9BF improved the MNG was calculated based on the predicted BF as inputs, the MNG accuracy decreased by 55% . Therefore, the integration of respiratory measurements for BF signals are also delayed during transitions and despite not having exactly the same dynamics as the In addition to O2 deficit at the on-transition phase is counter-balanced by the excess of O2 consumption during recoveryStudies that optimize the MNG) of the aerobic system based on the predicted ADL for ordinary people or patient populations, or during prescribed variations in work rate, such as athletic training.The ADL. Thus, the exercise protocols were limited to light and moderate activities with intensities lower than ~6 METs, and any attempt to extend this range should include extensive testing for reliability. Any studies that investigate the algorithm proposed in the current study for high intensity activities must recognize that ADL where intense activities are unlikely to occurThe purpose of the current study was to predict The population tested in the current study  had narrow weight and age ranges which might also restrict the use of the proposed algorithm. Further studies are necessary to verify the reliability of the MNG can be used to investigate aerobic response during realistic activities with direct applicability for the general population. Developing the aforementioned predictive model will provide a unique opportunity for continued lifelong In conclusion, oxygen consumption dynamics can be predicted from the fusion of data from non-intrusive wearable sensors and machine learning prediction algorithms. Longitudinal predictions of oxygen uptake can be obtained from wearables based on the validation completed in the current study for activities of daily living and random over-ground walking. The proposed random forest ensemble predictor in conjunction with How to cite this article: Beltrame, T. et al. Prediction of oxygen uptake dynamics by machine learning analysis of wearable sensors during activities of daily living. Sci. Rep.7, 45738; doi: 10.1038/srep45738 (2017).Publisher's note: Springer Nature remains neutral with regard to jurisdictional claims in published maps and institutional affiliations.


The objective of this study was to compare long-latency auditory evoked potentials before and after hearing aid fittings in children with sensorineural hearing loss compared with age-matched children with normal hearing.Thirty-two subjects of both genders aged 7 to 12 years participated in this study and were divided into two groups as follows: 14 children with normal hearing were assigned to the control group (mean age 9 years and 8 months), and 18 children with mild to moderate symmetrical bilateral sensorineural hearing loss were assigned to the study group (mean age 9 years and 2 months). The children underwent tympanometry, pure tone and speech audiometry and long-latency auditory evoked potential testing with speech and tone burst stimuli. The groups were assessed at three time points.The study group had a lower percentage of positive responses, lower P1-N1 and P2-N2 amplitudes (speech and tone burst), and increased latencies for the P1 and P300 components following the tone burst stimuli. They also showed improvements in long-latency auditory evoked potentials (with regard to both the amplitude and presence of responses) after hearing aid use.Alterations in the central auditory pathways can be identified using P1-N1 and P2-N2 amplitude components, and the presence of these components increases after a short period of auditory stimulation (hearing aid use). These findings emphasize the importance of using these amplitude components to monitor the neuroplasticity of the central auditory nervous system in hearing aid users. Hearing thresholds obtained using behavioral tests do not provide comprehensive data regarding the contribution of individual sound amplification devices (hearing aids [HAs]) and/or cochlear implants to the central auditory nervous system. For this reason, the use of objective tests is essential Currently, long-latency auditory evoked potentials (LLAEPs) are used to investigate impairments in the central auditory pathways of children LLAEPs are composed of the P1, N1, P2, N2 and P300 components. P1, N1 and P2 are considered exogenous components, i.e., they are influenced by the acoustic characteristics of a stimulus, while the N2 and P300 components are endogenous, i.e., they are most influenced by intrinsic events such as perception and cognition LLAEPs can be obtained using different acoustic stimuli (pure tones or speech), and responses to tonal stimuli have lower latency than those obtained from a speech stimulus Impairment of the LLAEP components P1, N1, P2, N2) in children with mild to moderate SNHL has been reported in a previous study, which showed that this population has a deficit in central auditory processing , N2 in cAccording to the literature, the presence of the P1, N1, P2 and N2 components using speech and tone burst stimuli in children with SNHL is positively correlated with the duration of HA use, and the latency of the N1 and P2 components is positively correlated with both the duration of HA use and the child’s age The P300 component is present among people with hearing loss as long as the individual can perceive and distinguish acoustic stimuli Individuals (children and adults) with severe or profound congenital hearing loss showed 58.6% presence of P300. Individuals with profound hearing loss showed lower amplitudes than those with severe hearing loss, but there were no differences in latency Given the importance of proper central auditory nervous system functioning in childhood development, the aim of this study was to provide scientific evidence for the development and plasticity of the central auditory nervous system in response to HA use using LLAEPs. The study will evaluate the effectiveness and benefits of sound amplification (stimulation) in hearing-impaired children. This study compares LLAEPs in children with SNHL before and after HA fittings compared with those in age-matched children with normal hearing.This was a longitudinal prospective clinical study approved by the Ethics Committee for Research Project Analysis under Protocol No 266512/2013. The participants’ legal guardians read and signed the Terms of Free and Informed Consent (TFIC) form, and the children signed the Terms of Assent form.A total of 32 subjects of both genders aged 7 to 12 years participated in this study. The participants were divided into two groups as follows: 14 comprised the control group , and 18 comprised the study group .The CG comprised children who had normal otoscopy findings, auditory thresholds up to 15 dB HL (frequencies of 250 to 8000 Hz), a type A tympanometric curve The SG comprised children who had normal otoscopy findings, mild to moderate bilateral symmetrical and flat SNHL ). All children with hearing loss were referred to the Auditory Health Clinic of the Clinics Hospital by the Central System for Regularization of Health Services Offering  of the State Health Department of São Paulo. Due to the above referral system, at the time of the audiological and electrophysiological hearing assessments, none of the children had undergone any type of rehabilitation for their hearing loss.Hearing loss in the SG was diagnosed at the Hearing Clinic of the Clinical Hospital of the School Medicine of the University of São Paulo - Division of Clinical Otorhinolaryngology , suspected prematurity in 5.55% 5, suspecAfter the children were diagnosed with hearing loss, they were referred for electrophysiological evaluation of hearing. At a later date, they returned to the outpatient clinic for selection and fitting of HAs.In all cases, the HA was a bilateral mini retroauricular-type device with digital technology and non-linear signal processing. All children with mild or moderate hearing loss underwent a HA fitting. The children used their HAs daily, and the daily use was monitored through the processing algorithm that is built into the HA’s memory (mean time of 8.5 hours of daily use).The children underwent tonal and vocal audiometry using a Grason Stadler GSI 61 audiometer and a TDH 50 supra-aural earphone, acoustic emittance measurements using a Madsen Model Zodiac 901 emittance meter, and LLAEP examination using a two-channel device  calibrated at listening level (dBHL). The acoustic stimulation used to acquire the LLAEPs was presented by a sound field system with speakers positioned at a 90-degree angle and 45 cm from the ear to be tested.Both groups were assessed at three different time points: baseline (M0), 3 months after the initial assessment (M3), and 9 months after the initial assessment (M9). The SG children did not use HAs at M0.The LLAEPs were obtained by the same evaluator at the three time points. The components were analyzed by the evaluator and two other professionals with experience in hearing electrophysiology using consensus scoring.To capture the P1, N1, P2, N2 and P300 components of the LLAEPs, the child was placed in a reclining chair in an acoustically treated room with appropriate electrical access. The skin was cleaned with abrasive paste, and the electrodes were attached to the skin with electrolytic paste and adhesive tape (Micropore) at the active vertex (Cz) and ground (Fpz) based on the International Electrode System standard IES 10-20 LLAEPs were evoked by tone burst stimuli using 1000 Hz as the frequent stimulus and 2000 Hz as the rare stimulus. The stimuli were delivered for 50 ms at 75 dBnHL at a display speed of 1.1 stimuli per second using an exact Blackman envelope and an interstimulus interval (ISI) of 860 ms. We chose the frequencies between 1000 and 2000 Hz based on the literature and used the protocol proposed by Hall, 2007.For the speech stimuli, the /ba/ syllable was used as the frequent stimulus and the /da/ syllable was used as the rare stimulus. The stimuli were presented at 75 dBnHL at a presentation rate of 1.1 stimuli per second. The frequent stimulus (/ba/) had a duration of 114 ms and an ISI of 799 ms, while the rare stimulus (/da/) had a duration of 206 ms and an ISI of 690 ms. Both stimuli were synthetic. The specific characteristics of the /ba/ and /da/ speech sounds are described in A total of 300 stimuli (15% rare and 85% frequent) were used to capture the P300 component. The children were instructed to pay attention to the rare stimuli, which occurred randomly among a series of frequent stimuli, and to raise their hand whenever the rare event occurred.During data collection, responses were recorded on two charts: one chart corresponded to the frequent stimulus and identified and analyzed the P1, N1, P2 and N2 components, and another chart corresponded to the rare stimulus and identified and analyzed the P300 component. The amplitude and latency of all components and the presence and absence of responses for each ear were analyzed.Each child was instructed to look at a fixed point two meters in front of him/her. The charts were accepted for analysis when a maximum of 30 artifacts were present.In addition to the tabulation of the latency and amplitude values of the LLAEP components at each assessment point, the LLAEP components were classified as present or absent for each individual and for each studied ear.First, the percentages of present and absent LLAEP components were calculated for both groups. We compared the latency and amplitude values within each group and between groups at each time point .The Shapiro-Wilk and Levene tests were performed to verify that the groups had a normal distribution and to determine whether there was homogeneity of variances between groups, respectively.p value ≤0.05) for all analyses.Descriptive analyses were performed using means and standard deviations (± SD). When comparing three values, the three-factor repeated measures analysis of variance was used. When comparing two values, the two-factor analysis or the Bonferroni test was used. The significance level was set at 5%  in the P1-N1 amplitude value in the right ear (RE), and the SG presented a lower mean amplitude at all assessment points , there was a statistically significant difference (t points .p=0.003), P2-N2 in the LE at M3 (p=0.032) and P300 at M0 (p=0.010)  .p=0.027; M0xM9 p=0.060) and in the P300 amplitude in the LE   .The comparison of the CG and SG at the three time points and the two-factor comparison revealed no statistically significant difference in the latency of the LLAEP components .p=0.007; M3XM9 p=0.010) and N2  were found for the LE in the SG. In the CG, a statistically significant increase in the P2 latency  in the LE was observed , statistically significant differences in the P1-N1 amplitude were found in both ears, and the SG had lower amplitudes at all time points .p=0.008) and in the LE at M1 (p=0.036). We also observed significant differences in P2-N2 in the RE at M3 (p=0.039) and M9 (p=0.019)  . For botComparisons between the time points revealed no statistically significant differences between the P1-N1, P2, N2 and P300 amplitudes in either the CG or SG .p=0.013) in the LE, with the SG showing a greater latency value.The comparison of the control and study groups at the three time points revealed no significant difference in the latency of the LLAEP components. In the two-factor comparison, we observed a significant difference only for P300 at M0  and P1 in the LE  and P300 in the RE  in the SG  components are influenced by the physical characteristics of the stimulus The comparison of the P300 amplitudes in both the CG and SG  and 5 reThese findings are similar to those reported in a study showing that P300 was present in 100% of subjects with normal hearing or mild to moderate SNHL at an intensity of 80 dB SPL Furthermore, although the difference was not statistically significant, the SG showed an improvement in the P300 amplitude between M0 and M3 for both stimulus types. These results suggest possible neuroplasticity due to HA use. According to the literature, neuroplasticity occurs as a result of the nervous system’s ability to reorganize its structures, functions and connections in response to intrinsic and extrinsic stimuli According to the literature In this study, the comparison of responses to the speech stimulus between the CG and SG  and 3 shThe results of this study show that, for most LLAEP components, the SG and CG had equivalent acoustic stimulus transmission/processing speeds. These findings may be related to the SG’s degree of hearing loss. It is noteworthy that in mild/moderate recruitment hearing loss, the impairment may reside in the outer hair cells (OHCs) Another possible cause of the similar latency found in both groups may be related to plasticity resulting from auditory deprivation, i.e., the central nervous system reorganizes to compensate for a peripheral receptor disorder Although there were no differences in latency between the two groups, the intra-group comparisons at the different time points revealed some differences  and 4. TThe results show that after three months of HA use, there were improvements in the response generation times of the P2 and N2 (speech stimulus) and P1 and P300 (tone burst stimulus) components, likely due to neuroplasticity in response to auditory stimulation (HA use).These findings are in accordance with a previous study There are some limitations to the current study, such as the number of individuals in the sample. The number of participants in the SG was variable at different time points due to the longitudinal design, which depended on the compliance of patients for audiological follow-up. For this reason, it was not possible to achieve an equal number of individuals at the three time points for audiological follow-up. In addition, it was not possible to obtain a homogeneous sample with consistent etiology and hearing loss onset. Regarding the control group, we believe that the low number of individuals may be related to the fact that they were typical developed children, with no hearing complaints.In conclusion, the results of this study demonstrated reductions in amplitudes of the P1-N1 and P2-N2 components of the LLAEP and a greater percentage of absent responses in children with mild to moderate SNHL, suggesting a deficit in the activation of the cortical structures responsible for auditory processing of the exogenous characteristics of these stimuli.P300 showed similar responses between the groups, suggesting that the endogenous potential was less affected than the exogenous potential by the sensory input deficiency caused by the hearing loss in this population.The latency of the LLAEP components showed little variability between the groups, indicating that sensory hearing loss did not interfere with the stimulus processing speed. The responses were steady across the three time points, demonstrating that this measurement is minimally affected over a short time interval (9 months).The results of the present study show that it is possible to identify changes in the central auditory pathway using P1-N1 and P2-N2 component amplitudes and that there is an increased response of these components to a short period of auditory stimulation . Therefore, the importance of using these components to monitor the neuroplasticity of the central auditory nervous system in HA users should be emphasized.Leite RA participated in the conception and design of the study, acquisition, analysis and interpretation of the data, drafting and revision of the manuscript for important intellectual content, and approval of the final version of the manuscript to be published. Magliaro FC participated in the conception and design of the study, acquisition, analysis and interpretation of the data, drafting and revision of the manuscript for important intellectual content, and approval of the final version of the manuscript to be published. Matas CG participated in the conception and design of the study, the analysis and interpretation of the data, drafting and revision of the manuscript for important intellectual content, and approval of the final version of the manuscript to be published. Raimundo JC participated in the conception and design of the study, acquisition of data, and critical revision of the manuscript for important intellectual content. Bento RF participated in critically revising the manuscript for important intellectual content.


PTPRQ gene, encoding protein tyrosine phosphatase receptor Q, is essential for the normal maturation and function of hair bundle in the cochlea. Its mutations can cause the defects of stereocilia in hair cell, which lead to nonsyndromic sensorineural hearing loss. Using next-generation sequencing and Sanger sequencing method, we identified a novel compound heterozygous missense mutation, c.4472C>T p.T1491M  and c.1973T>C p.V658A , in PTPRQ gene. The two mutations are the first reported to be the cause of recessively inherited sensorineural hearing loss. Hearing loss levels and progression involved by PTPRQ mutations among the existing cases seem to be varied, and the relationship between genotypes and phenotypes is unclear. Our data here further prove the important role of PTPRQ in auditory function and provide more information for the further mechanism research of PTPRQ-related hearing loss. Genetic factors are the major cause of congenital sensorineural hearing loss (SNHL). Approximately 80% of nonsyndromic genetic hearing loss is autosomal recessive inheritance [http://hereditaryhearingloss.org).Hearing loss is one of the most common sensory disabilities in humans. According to the latest data of WHO, there are 360 million people—over 5% of the world's population—suffering from hearing loss, with 32 million are children  was performed after the patient underwent otoscopic examination in our department. Degree of hearing loss was assessed by using pure tones. The stimuli were produced in the frequencies of 0.25, 0.5, 1, 2, 4, and 6 kHz. By using the stimulus-reply-visual reinforcement conditioning, the minimum response level was obtained in the lowest intensity which the child responded.Peripheral venous blood samples from all the family members were obtained for genetic analysis. Genomic DNA was extracted from the blood samples using QIAamp DSP DNA Blood Mini Kit  according to kit's protocol.The target deafness-related gene capture and next-generation sequencing + Sanger sequencing were performed by MyGenostics Inc. . First, the genomic DNA was fragmented to special size about 350–400 base pair for library construction. End-repair and Illumina adapter ligation were taken according to the Illumina protocols. After PCR amplification, target DNA fragments were captured with biotinylated single-strand DNA capture probe  by hybridization. The target gene fragments were enriched, and then high-throughput sequencing was performed using Illumina HiSeq2000 Analyzer for automated cycles per read. Primary data were generated using Trim-Galore software (version 0.4.3). Reads were matched to NCBI37/hg19 using BWA program. Previously identified SNPs were annotated using CCDS, human genome database (HG19), and dbSNP (v138). SIFT and POLYPHEN2 were utilized to predict the function of SNP-affected protein.http://www.swissmodel.expasy.org/). The complete protein sequence of human PTPRQ is available in the NCBI GenBank (NP_001138498.1). Data were showed by JavaScript Protein Viewer.The molecular homology modeling of the human wild type and mutations was built up by SWISS-MODEL (http://sift.jcvi.org/) and POLYPHEN2 (http://genetics.bwh.harvard.edu/pph2/) were used to analyze the amino acid substitutions of p.T1491M and p.V658A. Both programs predicted these two mutations to be deleterious, which means they probably damage and affect protein functions. The sequencing results showed that the two parents were heterozygous carriers of c.4472C>T  and c.1973T>C , which demonstrated the compound heterozygous cosegregating mutation with the phenotype in II1 , these mutations corresponded to c.4472C>T and c.1973T>C, occurring in exon26 and exon13 of PTPRQ. The c.4472C>T leads to a single substitution within the fibronectin type III (FNIII) domain, from threonine to methionine (p.T1491M), and the other, c.1973T>C, leads to a single substitution  within the FNIII domain. SIFT (e in II1 . The frehttp://www.swissmodel.expasy.or-g/), which predict the sequence homology. The p.T1491M protein model, covering the target sequence (residues 1163–1564), was constructed based on the receptor-type protein tyrosine phosphatase S (PDB ID: 4pbx.1.A). Sequence identity between the target and template was 25.78%. As shown in Protein tertiary structures were modelling with SWISS-MODEL  showed that reproducible wave cannot be elicited at 105 dBnHL bilaterally. The diagnosis of congenital severe-profound sensorineural hearing loss (SNHL) was made. Her parents took her for the hearing compensation in our hospital after four months. A visual reinforcement audiometry (VRA), computed tomography (CT) scan of temporal bone, and magnetic resonance (MR) imaging were performed. The VRA showed no response of both ears in all frequencies . The CT Hair cells are the essential sound receptors of auditory system. The hair bundle, comprised of about 100 actin-filled stereocilia, on the top of hair cells can help the sound transduction from mechanical signal to electrical signal. PTPRQ is a stereociliar membrane protein which belongs to the type III receptor-like protein tyrosine phosphatase (PTPase). It has three domains: the fibronectin type III- (FNIII-) like extracellular domain, the transmembrane domain, and the cytosolic PTPase domain, which have phosphatidylinositol phosphatase activity . The PTPThere are 9 mutation variants of PTPRQ gene causing SNHL reported until now. They have been identified in different ethnic groups and countries, including c.1285C>T in Palestinian ; c.1491T−/− mice showed defects in the hair bundles of the saccule and ampullae [In this study, the patient II1 did not pass the examinations of newborn hearing screening when she was 42 days. In subsequent hearing tests, she went on to exhibit to a series of severe-profound sensorineural hearing loss symptoms. In the past studies, the affected patients had different hearing levels from moderate to profound and different progressions from stable to progressive. In our case, the patient had a severe-profound degree of hearing loss and a stable progression. Based on the results of existing PTPRQ mutation reports, we suspect that there is no obvious correlation between genotype and phenotype. PTPRQ mutation appeared to be liable for the vestibular dysfunction. The patients with PTPRQ mutations always had vertigo or dizziness. Consistent with this manifestation, the Ptprqampullae . Howeverampullae . When paampullae . We specIn summary, our findings suggest that the novel compound heterozygous PTPRQ mutations, c.4472C>T (p.T1491M) and c.1973T>C (p.V658A), are the cause of congenital SNHL in this family. The identification of additional mutations here further confirms the key role of PTPRQ in hearing function. More precise mechanism researches are needed for a better understanding of the gene.


Perfluorocarbon liquid (PFCL) is widely used as an intraoperative heavy tamponade to flatten the retina and is replaced with silicone oil (SO) at the end of the surgery. Due to the long tamponade period, the SO is known to remain attached to the retina at the time of removal, and is commonly termed “sticky oil”. The aim of this present study was to report a case of SO stickily attached to the retina via PFCL without tamponade period.A 39-year-old male was referred to our hospital due to decreased vision and visual field defect in his right eye. Upon examination, he was diagnosed with rhegmatogenous retinal detachment in that eye. For treatment, he underwent vitrectomy with the use of PFCL and SO. The direct exchange of PFCL with SO resulted in residual subretinal fluid, so we subsequently attempted to remove the SO. However, a SO bubble adhering to the PFCL was visible on the posterior pole. After aspiration of the PFCL beneath the sticky SO, the SO was easily separated and removed from the retina.Our findings show that SO can become tightly adhered to the retinal surface via PFCL during vitrectomy, and that the sticky SO can be safely removed via aspiration of the PFCL layer underneath the SO. Over the past few decades, silicone oil (SO) has been widely used in vitreoretinal surgery as a long-term tamponade in the surgical treatment of patients with retinal detachment . PerfluoIn this study, we present a case in which SO became adhered to the surface of the retinal via PFCL during vitrectomy.A 39-year-old male was referred to our hospital due to decreased vision and visual field defect in his right eye. Upon examination, this right-eye visual acuity was 0.03 and bullous rhegmatogenous retinal detachment was found in three quadrants, yet not in the upper part of the retina Fig. . For treThe PFCL layer between the SO and the underlying retina was easily aspirated and the SO was released from the retinal surface. Post release, the SO was relocated to the anterior chamber and safely removed through a corneal limbal incision. After complete removal of the SO, pneumatic retinal replacement and laser photocoagulation were performed, and sulfur hexafluoride gas was used as a temporary intraocular tamponade. At 7-months postoperative, the patient’s retina remained attached and his corrected visual acuity improved to 0.7.We performed an in vitro experiment to investigate the interaction between PFCL and SO. For that experiment, a glass test-tube was filled with balanced salt solution (BSS). Next, 0.2 ml of PFCL was introduced into the solution and placed onto the bottom of the tube Fig. . Next, 0In this study, we report a case in which an SO bubble became tightly adhered to the retinal surface during vitrectomy. To the best of our knowledge, this is the first report of sticky SO becoming tightly adhered to the retinal surface via PFCL. The difficulty of removing SO at the end of the tamponade period has previously been reported. In a retrospective study, Veckeneer et al. reported 28 of 234 cases (12.0%) in which patches of sticky SO remnants adhered to the retina within the posterior pole during removal of the SO . Sleep eIt should be noted that the water layer between PFCL and SO can flow into the subretinal space through the retina tear during the direct exchange of PFCL with SO. To avoid such a phenomenon, it is recommended that the aqueous interface be removed in the early stage of the exchange. Complete removal of aqueous liquid can result in a direct contact between PFCL and SO. Subsequently, these two substances can become adhered to each other due to the nonallele nature of the two phases . In thisIn our in vitro experiment, the three components  were found to form their shapes to reach the point where their total energy would be lowest, and we found that the SO fully covered the surface of PFCL. To separate SO from PFCL, the applied energy must exceed the adhesion force between the SO and the PFCL. Dresp et al. and Ghoraba et al. reported that the cohesion of SO is lower than the adhesion forces, and concluded that aspiration of SO from the vitreous cavity after the direct exchange of PFCL with SO can be extremely difficult , 9.Microincision vitreous surgery is gaining popularity, and is being applied to complicated cases more than ever before. Due to the small diameter of available cannula devices, high aspiration forces are necessary to remove high-viscosity materials such as SO. To remove sticky SO droplets, the tip of the cannula should be placed close to the retinal surface. It is known that strong suction forces adjacent to the retina can lead to the development of an iatrogenic retinal tear. In contrast to SO, PFCL can be removed via the use of a relatively small suction force. In our case, SO was easily removed from the areas of the retina that were coated with PFCL by aspirating the PFCL layer beneath the SO.Our findings show that in cases involving a tight adhesion of SO on the surface of the retina during microincision vitreous surgery, the SO bubble can be easily removed by aspiration of the PFCL layer underneath the SO.


BBB) functions, there have been no investigations regarding the blood–nerve barrier (BNB). In this study, we examine how fingolimod affects the BNB.The main effect of fingolimod is thought to be functional antagonism of lymphocytic S1P1 receptors and the prevention of lymphocyte egress from lymphoid tissues, thereby reducing lymphocyte infiltration into the nervous system. However, a growing number of reports suggest that fingolimod also has a direct effect on several cell types in the nervous system. Although we previously reported that fingolimod enhances blood–brain barrier (HPnMEC) was used to evaluate BNB barrier properties. We examined tight junction proteins and barrier functions of HPnMECs in conditioned medium with or without fingolimod‐phosphate and blood sera from patients with typical chronic inflammatory demyelinating polyneuropathy (CIDP).An immortalized human peripheral nerve microvascular endothelial cell line (mRNA and protein as well as TEER values in HPnMECs. Conversely, typical CIDP sera decreased claudin‐5 mRNA/protein levels and TEER values in HPnMECs; however, pretreatment with fingolimod‐phosphate inhibited the effects of the typical CIDP sera.Incubation with fingolimod‐phosphate increased levels of claudin‐5 BNB and enhances barrier properties. This mechanism may be a viable therapeutic target for CIDP, and fingolimod may be useful in patients with typical CIDP who have severe barrier disruption.Fingolimod‐phosphate directly modifies the  However, there has also been some focus on its multifunctional effects on several cell types such as astrocytes, oligodendrocytes, neurons, and microglia  as previously described  at 37°C. Solute paracellular permeability was assessed using 10 kDa dextran‐conjugated FITC , and fluorescence in the lower chamber was measured using an MX3000P instrument (Stratagene) as previously described , and a two‐sided p value <.05 was considered to be statistically significant.All comparisons of the median values between the groups were made using the Mann–Whitney 33.1We examined which S1P receptors were expressed by the HPnMECs using RT‐PCR. HPnMECs expressed the S1P1 and S1P2 receptors , a previous report showed that fingolimod ameliorates disease conditions in animal models of experimental autoimmune neuritis (Zhang, Zhang, Fauser, & Schluesener, An increasing number of studies have stressed that fingolimod also has a direct effect on several cells within the CNS, including astrocytes, oligodendrocytes, microglia, and neurons (Miron, Schubart, & Antel, There are many phenotypic variants of CIDP suggesting that it may not be a discrete disease and that the pathogenic mechanism of CIDP is heterogeneous (Mathey et al., In conclusion, the present findings demonstrate that pretreatment with fingolimod‐phosphate enhances the barrier properties of the BNB by upregulating claudin‐5 expression in HPnMECs. These results suggest that fingolimod may prevent humeral factors from crossing the BNB into the PNS via its direct effects on HPnMECs. The direct BNB‐modulating effects of fingolimod may represent a possible novel venue for treating patients with CIDP.None declared.


It is shown that the unstable evolutions of the Hermite-Gauss-type stationary solutions for the nonlocal nonlinear Schrödinger equation with the exponential-decay response function can evolve into chaotic states. This new kind of entities are referred to as chaoticons because they exhibit not only chaotic properties  but also soliton-like properties (with invariant statistic width and interaction of quasi-elastic collisions). They have been demonstrated in a large variety of physical systems, including optics, hydrodynamics, particle physics, electrical circuits and even astrophysics1245678910R(x) is the (nonlinear) response function, which must be symmetry for the existence of the soliton-like solutionswhere the real positive function x, q at that point but also by the wave in its vicinity. This kind of nonlocal nonlinear response has been found in several systems, such as Bose-Einstein condensates3115V1314R(x) = δ(x) in 222324257181920181911V can be simplified to a quadratic form in the limit of strong nonlocality, while the latter that can describe physically real materials is singular and the corresponding NNLSE cannot be generally reduced to a linear Snyder-Mitchell modeThe nonlocal nonlinearity  in Unlike the  nonlinear Schrödinger equation, which is integrable and can be solved via the inverse scattering transform method193637In this letter, we investigate the evolution of the (1 + 1)-dimensional NNLSE with the exponential-decay response function for the initial inputs of the HGT stationary solutions. As has been mentioned39We consider here the NNLSE  with the132022x = 0. This case corresponds to the model for the propagation of the (1 + 1)-dimensional paraxial optical beam in nematic liquid crystals, which can be described by the coupled partial differential equations10111516which has a singularity at q is the dimensionless slowly-varying complex amplitude of the optical field, x and “time” t stand for, respectively, the dimensionless transverse coordinate and the dimensionless propagation direction coordinate, the “potential” −V represents the nonlinear induced refractive index. Obviously, the set of wm to the statistic width of the wave w denotes the degree of nonlocality13w is defined by the second-order moment wm/w, the stronger the nonlocality.where The NNLSE  permits 192021uN is a real function and bN is a real constant. It was numerically found that in the case with the exponential-decay response function uN(x) is of N-humps  HGT-structure20u1(x) has a single-hump Gauss-type shape. It was also proved that uN(x)(N ≥ 2) can exist only when the parameters wm and bN satisfy where q = uN(x) by means of the split-step methodwm = 10 and w(0) = 1 unless otherwise stated] is considered. The unstable evolutionN > 4)are given in N = 7 and 12 are displayed without loss of generality. It is clear that the profiles starting from regular multi-humps turn to be irregular shapes, several of which are shown in We simulate Since a positive Lyapunov exponent is a signature of chaos, we explore the maximal Lyapunov exponent363738q1 and q2 in the Hilbert space (the L2 norm in the Hilbert space), the two initial values q1 = uN(x) and q2 = uN(x) + r(x), and r(x) is a random perturbation function .where MM = 2048, 4096, 8192, each of them with the window size L = 40, 50, 60. We find that the statistical errors of the maximal Lyapunov exponents for every initial input are less than 10%  or the renormalization step-size). Then we get the average values of the maximal Lyapunov exponents for the HGT stationary solutions with N ≤ 12, as summarized in N, while those for solitons are equal to zero. The occurrence of chaos can be understood as a consequence of the complex interactions among humps. The more humps the profile possesses, the more complex the interactions are, which leads to a higher degree of the chaos.For a partial differential equation, the Lyapunov spectrum  converges to a smooth curve, although the number of Lyapunov exponents is dependent on the number of discretization k is a positive constant) and It is especially important to make clear that the chaotic phenomenon described above is due to the intrinsic nature of the system but not numerically induced chaos45Eq. (1)−9. It will make sense that the regular profiles of the initial HGT stationary solutions will be considered to become completely irregular once the deviation reaches the order of 1. We can, therefore, estimate tc  by tc ≈ 20.7/λ, as shown in Next, we will prove that the maximal Lyapunov exponents coincide with the growth rates of the initial numerical errors. Although, literally, the maximal Lyapunov exponent measures the typical exponential rate of growth of an infinitesimal perturbation, the growth of a noninfinitesimal deviation is usually well described in this way. The numerical error of the HGT stationary solutions computed by the Newton iteration method in double precision is assumed to be of the order of 10s(0) = 0, since |uN(x)|2 is symmetric. s for the HGT stationary solutions with N = 7 and 12 in the time interval  and , respectively. It can be seen that |s| starts from the close neighbour of zero and then rises abruptly around a certain tcs, which is defined as tcs represents the critical time of becoming completely irregular attained from the direct statistic method. The comparison between tc and tcs is given in Obviously, there is ξ and ηIn addition, it is expected that the spatial patterns will be spatially decorrelated in a system described by a partial-differential evolution equation with temporally chaotic behavior505152c for the HGT stationary solutions are depicted in c| equals 1 along the line ξ = η and decreases rapidly with the separation of two locations. The quick drop of correlation in the x direction means the spatial decoherence52where the superscript * denotes the conjugate complex. The modulus of w, as shown in Chaotic as they are, the evolution of the unstable HGT stationary solutions maintain almost invariant statistic width w) to prevent the overlap of waves, and for each case of different Ns, both of the initial chaoticons are q for t0 ≥ tc, which means that the inputs are completely irregular states. We can observe that the two chaoticons attract each other, and then combine and separate quasi-periodically, much like elastic collisions between two particles. In fact, they will eventually fuse together accompanied by small energy loss to radiation after a much longer evolution. Hence, strictly speaking, the interaction is quasi-elastic.Amongst all soliton properties, the important fascinating one is the particle-like interaction78wm for every HGT stationary solution with a given w. It means that the HGT stationary solutions in stronger nonlocality will evolve longer before they break up. Therefore, the radiation waves are believed to be absent in a strongly nonlocal nonlinear case27Firstly, it is worth underlining that the evolution of the unstable stationary solutions in generally or weakly nonlocal nonlinearityWe have found that the unstable evolution of the (1 + 1)-dimensional NNLSE with the exponential-decay response function for the initial inputs of the HGT stationary solutions will evolve into a new kind of chaoticon, which occur only in the case of strongly nonlocal nonlinearity. The chaoticon exhibits both chaotic and soliton-like properties. The chaotic behavior is signified by the positive maximal Lyapunov exponents and spatial decoherence. The soliton-like property is demonstrated by the invariant statistic width during the evolution, as well as the quasi-elastic collisions during the interaction.How to cite this article: Zhong, L. et al. Chaoticons described by nonlocal nonlinear Schrödinger equation. Sci. Rep.7, 41438; doi: 10.1038/srep41438 (2017).Publisher's note: Springer Nature remains neutral with regard to jurisdictional claims in published maps and institutional affiliations.


Retinopathy of prematurity (ROP) is an ocular disorder which affects infants born before 34 weeks of gestation and/or with birth weight of less than 2000 grams. If not detected on time and appropriately managed, it can lead to irreversible blindness.The retinal blood vessels first appear between 15–18 weeks of gestation. These vessels grow outwards from the central part of the retina and extend towards the retinal periphery. The nasal part of the retina is fully vascularised by 36 weeks of gestation followed by the temporal retina which is completely vascularised between 36–40 weeks of gestation age . FollowiThe development of ROP can be divided into two phases - an initial phase of delayed growth of retinal vessels followed by a second phase of retinal vessel proliferation .At birth the lungs of an infant born preterm are immature placing him/her at a high risk of developing abnormally low level of oxygen in arterial blood.To overcome this, the newborn infant is given supplemental oxygen in NICU. Prior to 32 weeks of gestation, the retina is very immature and the retinal metabolic demand is low. This excess oxygen creates retinal hyperoxia and oxygen toxicity, inhibiting the production of VEGF. This is followed by temporary stopping or stoppage of normal retinal growth, and constriction of new immature vessels. As a result, there is a reduction of blood supply to retinal tissue and shortage of oxygen needed for metabolism.With increasing age of the preterm infant the retina matures. There is an increase in metabolic demand and oxygen consumption by the retina, creating a relative decrease in oxygen level. This promotes increase in the level of vascular endothelial growth factor (VEGF), triggering the formation of new blood vessels along the inner retinal surface. A demarcation ridge develops along the retina that separates the central vascularised retina from the peripheral avascular retina .The growth of retinal blood vessels at this stage may restart normally or may progress to significant ROP as seen by an abnormal growth of retinal vessels into the vitreous and over the surface of the retina. These new vessels are weak and underdeveloped failing to fulfill the oxygen demand of retinal tissue resulting in continuous growth of abnormal vessels. There is leakage of fluid or blood from these weak blood vessels. If not treated on time this can result in scarring or traction of the retina leading to retinal detachment and blindness .


Congenital middle ear malformations are rare. Most part of them are usually associated with other malformations, such as aural atresia, microtia, and dysmorphic craniofacial features. A clinical case of a 24-year-old male with a right-sided conductive hearing loss since his childhood, without craniofacial malformation, is presented. He was proposed for exploratory tympanotomy under the suspicious diagnosis of otosclerosis. The surgery revealed an abnormal location of stapes' superstructure, which was attached to the promontory and had an isolated and mobile osseous footplate in the oval window. A stapes prosthesis was inserted and resulted in closure of the air-bone gap by 25 dB. A review of the literature was also performed using MEDLINE. Two theories diverge on the embryologic origin of the stapes. Our findings seem to be in favour of the theory that defines two different embryologic origins to the stapes. Congenital ossicular chain malformations are present in less than 1 per 15.000 births [Minor abnormalities appear with a normal tympanic membrane, EAC, and pinna and may involve fixations, defects, or absence of middle ear structures . These minor middle ear abnormalities can appear to be isolated or in combination, involving more than one structure , 3.In middle ear abnormalities, patients classically present a nonprogressive conductive hearing loss and their most frequent problems are related to stapes ankylosis, discontinuity of the incudostapedial complex, or congenital absence of the stapes . OssiculThis is the report of a clinical case that may contribute to our knowledge of the stapes development.A 24-year-old healthy male was referred to Ear Nose and Throat consultation by long standing right ear hearing loss. He had no history of middle ear infection, neither of head injury, or even family history of hearing loss. In physical examination, he had no dysmorphic craniofacial features, presenting a normal pinna, EAC, and tympanic membrane. The otoscopy did not reveal any alteration; Rinne test was negative on the right side and neurological examination was normal.Pure tone audiometry proved a moderate conductive hearing loss in the right ear, with a 4-frequency  mean of 33 dB, and borderline normal thresholds in the left ear . TympanoThe possibility of being an otosclerosis was pondered and so an exploratory tympanotomy was performed. During the procedure a congenital stapes abnormality was detected. The stapes' superstructure was well formed with two limbs and head, inferiorly located, separated from the footplate, and attached to the promontory, as seen in The stapes' superstructure was removed from the promontory and then a stapedotomy was performed, creating a small fenestra in the centre of the footplate, in spite of being a mobile footplate. A fluoroplastic piston prosthesis was carefully placed see  and the The postoperative audiogram, The ossicular chain is formed by three ossicles, from lateral to medial: the malleus, the incus, and the stapes. Mechanical vibrations are transmitted into the inner ear structures via the integration of the stapes footplate and annular ligament into the oval window. Defects in the insertion of the stapes in the oval window produce a conductive hearing loss. A nonprogressive conductive hearing loss, in the range of 40 to 60 dB, with a normal tympanic membrane and no history of trauma or infection, is highly suggestive of a congenital ossicular malformation. As the preoperative diagnosis of congenital conductive hearing loss is challenging, an exploratory tympanotomy is an effective diagnostic method.The development of the middle ear's ossicles, in humans, is not yet totally understood. This way, there are conflicting opinions regarding the stapes origin, namely, about the possible contribution of the otic capsule to the stapedial footplate. Two main theories have been proposed to demonstrate the stapes origin. The first theory, described in 1942 by Cauldwell and Anson and later confirmed by Masuda et al. 1978), hypothesizes that the stapes has a dual source. The superstructure and the tympanic part of the footplate develop from Reichert's cartilage  and the vestibular part of the footplate develops from the otic capsule, thus both parts developing independently and later merging –7.8, hypothOn the other hand, several authors suggest that Reichert's cartilage is the common and unique origin of these structures. In 2003 Louryan et al. presented a case report of a unilateral congenital stapes misplacement, revealed by CT, without any relationship with the otic capsule; hence they assumed that the otic capsule is not mandatory for stapes development . HoweverIn the present clinical case, a complete isolated stapes' superstructure misplacement was observed in the promontory, while having a mobile osseous footplate in the oval window. Stapes' crura were attached to the promontory by a thin bony layer. These findings may suggest a dual origin of the stapes, as supported by Cauldwell and Anson theory, of a dual origin to the stapes' footplate. Whittmore, in 2013, and later Kanona, in 2015, described similar cases of stapes' superstructure misplaced inferiorly and with a mobile footplate in the absence of trauma . NeverthThe Teunissen and Cremers classification of congenital anomalies of the middle ear is the most generally accepted . This clCongenital minor abnormalities of the middle ear are rare causes of nonprogressive conductive hearing loss. Discontinuity between the stapes' superstructure and footplate was observed in this case report. The stapes' superstructure was attached to the promontory by a thin bony layer and totally disconnected from a thin footplate. There was no incudostapedial joint, only the presence of mucosal folds suspending between lenticular process of the incus and the head of the stapes. The footplate was mobile in the oval window. These findings seem to be in favour of the theory that defines two different embryologic origins to the stapes. Nevertheless, this is an area under actual investigation and further developments are expected.


Speech-in-noise tests have become a valuable part of the audiometric test battery providing an indication of a listener’s ability to function in background noise. A simple digits-in-noise (DIN) test could be valuable to support diagnostic hearing assessments, hearing aid fittings and counselling for both paediatric and adult populations.The objective of this study was to evaluate the South African English smartphone DIN test’s performance as part of the audiometric test battery.This descriptive study evaluated 109 adult subjects  with and without sensorineural hearing loss by comparing pure-tone air conduction thresholds, speech recognition monaural performance scores (SRS dB) and the DIN speech reception threshold (SRT). An additional nine adult hearing aid users  were included in a subset to determine aided and unaided DIN SRTs.sr = 0.81) and maximum SRS dB (r = 0.72). The DIN test had high sensitivity and specificity to identify abnormal pure-tone  and SRS dB  results. There was a mean signal-to-noise ratio (SNR) improvement in the aided condition that demonstrated an overall benefit of 0.84 SNR dB.The DIN SRT is strongly associated with the best ear 4 frequency pure-tone average (4FPTA) (The DIN SRT was significantly correlated with the best ear 4FPTA and maximum SRS dB. The DIN SRT provides a useful measure of speech recognition in noise that can evaluate hearing aid fittings, manage counselling and hearing expectations. One of the major problems persons with hearing loss experience is communication in the presence of background noise Taylor, . AmplifiBy the late 1970s, speech-in-noise tests became popular as a result of the pioneering work by Plomp and Mimpen  with theToday speech-in-noise tests are primarily used in the clinical setting to determine a person’s ability to function in a general communication environment by evaluating the speech understanding handicap caused by the hearing loss  was developed for diagnostic and clinical purposes ], its suitability for clinical use in an audiology clinic required investigation. Smits et al.  reportedGiven the potential benefit of a diagnostic version of the South African DIN, this study aimed to compare the DIN alongside standard diagnostic audiology tests in clinical practice. A comparison was conducted between pure-tone audiometry, speech recognition monaural performance scores (SRS dB) and the DIN SRT. The DIN was also used in a subset of participants to explore the potential benefit for use with hearing aid listeners by determining aided and unaided DIN SRTs.The institutional review board of the University of Pretoria approved the research study before data collection commenced.A comparative and correlational descriptive research design was followed. Various audiometric practices in the Gauteng region assisted with data collection. Each audiologist was supplied with a smartphone to conduct the South African English smartphone-based DIN on subjects in their own private practice or at the public health hospital audiology clinic. A convenience non-probability sampling procedure was followed with selected subjects as they were available and willing to volunteer to take part in the research study at clinical data collection sites. The subjects were assessed with a comprehensive audiometric test battery and the smartphone DIN in a single test session lasting approximately 1 h.An otoscopic evaluation was performed to allow observation of any obstruction in the external auditory meatus. Any ear canal obstructions were removed by a qualified audiologist or healthcare provider before testing commenced.A variety of clinical audiometers were used to conduct pure-tone air conduction, bone conduction and speech recognition testing. Air conduction and speech audiometry were done in a soundproof booth using supra-aural or insert earphones. A bone oscillator was used to conduct bone conduction audiometry. Air conduction thresholds across octave frequencies from 250 to 8000 Hz were determined, while bone conduction thresholds were determined from 500 to 4000 Hz. The modified Hughson–Westlake method was used to seek pure-tone air and bone conduction thresholds  to Afrikaans-speaking subjects . English additional language speakers could choose whether they would like speech recognition testing to be presented in Afrikaans or English. Normal maximum SRS dB was classified as a 100% word discrimination score at intensities ≤40 dB HL. The best ear maximum SRS dB was used in the analysis. The 50% SRS dB was not available for all subjects. Various sites were involved in data collection, thereby not using the same methods for determining SRS dB scores.Speech recognition testing was presented with live voice in Afrikaans or English. Speech recognition monaural performance scores were obtained across intensities by administering the The South African smartphone DIN App instructed the subject to enter his or her gender, date of birth, initials and surname. The subject placed the smartphone earphone set into the ears and listened to digits being repeated. A scroll bar allowed the subject to adjust the volume of the digits being repeated to a comfortable listening intensity. A ‘start test’ button presented the test. The subject entered the digit responses on the smartphone keypad.Once the DIN hearing screening test started, the test operates by varying the noise intensity level while having a fixed speech level when triplets with negative SNRs are presented. When triplets with positive SNRs are presented, the speech level becomes fixed and the noise level varies. The noise starts 500 ms before triplet presentation and stops 500 ms after triplet presentation. The digits were pronounced by a female speaker with natural intonation, for example, 6–9–0, spoken as six–nine–zero. The first digit-triplet set was presented at the subject’s comfortable listening intensity. The subject responded to the triplet set by entering the digit-triplet set on a pop-up keypad. The next digit is presented 2 dB higher (incorrect response) or 2 dB lower (correct response) based on the subject’s response.A subgroup of nine hearing aid users were asked to perform an additional DIN using their hearing aids. The DIN was presented using free-field speakers in a soundproof booth. The smartphone were connected to the free-field speakers. The subjects were seated 1 m from the speaker facing the speaker at 0°. The DIN was operated using the smartphone App and the subjects entered their response using the smartphone App.A Spearman correlation coefficient was determined to assess the relationship between the best ear 4FPTA, maximum SRS dB and the DIN SRT (1% significance level used).The performance of the South African English smartphone DIN was determined by comparing the SRT to the best ear 4FPTA and maximum SRS dB. Descriptive statistics, frequencies and proportions were determined for these variables. The sensitivity and specificity, positive predictive value (PPV) and negative predictive value (NPV) were calculated as an indication of the accuracy of the method for determining a hearing loss. Additionally, the area under the curve (AUROC) from a receiver operation characteristic curve (ROC) analysis was determined to provide a further indication of the accuracy between the variables.Descriptive statistics for aided and unaided hearing aid DIN SRTs were determined for a subset of nine subjects.A total of 109 adult subjects  participated in this study. The mean age was 55 years (20 SD) with a range of 16–89 years. The sample included 50 normal-hearing subjects and 59 subjects with a hearing loss. An additional subset of nine adult subjects  with an average age of 72 years  participated in this sub-study.r = 0.87; n = 111; p < 0.001), and the best ear 4FPTA and DIN SRT  and the maximum SRS dB and DIN SRT . The strongest correlation was between the best ear 4FPTA and maximum SRS dB and the weakest correlation was between the maximum SRS dB and the DIN SRT. A comparison between the best ear 4FPTA  and SRS dB  showed a significant difference between the two variables.Significant correlations  were evi DIN SRT   and unaided hearing aid DIN SRTs  showed a small increase in SRT with hearing aids . There was significant individual variability between subjects in the aided condition (−3.2 to −9.4 range) and unaided condition (−2 to −9.4 range) .An English smartphone DIN was successfully developed in 2016 to provide widespread hearing screening in South Africa  in line with previous results reported for the Dutch (r = 0.72), French (r = 0.77) and American English (r = 0.74) landline telephone DIN hearing screening tests  and corresponded to previous results comparing the Northwestern University Auditory Test No. 6 in quiet to the Words-in-Noise Test (Rs = 0.61)  related well to the Dutch  and American English  landline DINs  of hearing aid users were evaluated with the DIN with and without hearing aid amplification. The mean SNR improved in the aided condition and demonstrated an overall benefit of 0.84 SNR dB. There was significant individual variability between subjects in the aided condition (−3.2 to −9.4 SNR dB) and unaided condition (−2 to −9.4 SNR dB). The DIN can be valuable in a clinical audiology setting to provide individualised performance measures for hearing aid users in background noise (Smits et al., A small sample (The DIN SRT is strongly associated with the best ear 4FPTA and maximum SRS dB and could therefore provide complementary information on speech recognition impairment in noise. The DIN had high sensitivity and specificity to identify abnormal pure-tone and SRS dB results. The DIN SRT and best ear 4FPTA were significantly correlated with previously developed landline telephone DINs. The DIN SRT can also demonstrate benefit for hearing aid fittings. The test is quick to administer, inexpensive, readily available and provides information on the SNR loss. The DIN SRT could therefore be used as a counselling tool to evaluate hearing aid fittings, manage counselling and hearing expectations.