Fmr1 gene coding for the FMRP protein, which, with its paralogs FXR1 and FXR2, constitute a well-conserved family of RNA-binding proteins. Drosophila melanogaster is a good model for the syndrome because it has a unique fragile X-related gene: dFmr1. Recently, in addition to its confirmed role in the miRNA pathway, a function for dFmr1 in the piRNA pathway, operating in Drosophila gonads, has been established. In this review we report a summary of the piRNA pathways occurring in gonads with a special emphasis on the relationship between the piRNA genes and the crystal-Stellate system; we also analyze the roles of dFmr1 in the Drosophila gonads, exploring their genetic and biochemical interactions to reveal some unexpected connections.Fragile-X syndrome is the most common form of inherited mental retardation accompanied by other phenotypes, including macroorchidism. The disorder originates with mutations in the  Humans have three homologous genes coding for the Fragile-X Mental Retardation Proteins: Fmr1, FXR1, FXR2, with high sequence similarity; all of them code for RNA-binding proteins [Fmr1, located on the X chromosome and coding for FMRP proteins, are responsible for the syndrome. FMRP and its autosomal paralogs, the Fragile X-Related proteins FXR1P and FXR2P, constitute a conserved, small family of RNA-binding proteins. FMRP has been studied in humans, mice, and Drosophila, principally in relation to its role in the nervous system, at the synapses, as a translational regulator working with different hypothesized mechanisms: (i) by repressing translational initiation [proteins . Mutatioitiation ,5,6;  are homologous repetitive sequences located mainly at three different positions on the sexual chromosomes: Stellate repeats are located on the X chromosome, at the euchromatic 12E region and at the heterochromatic h26 region; crystal is on the Y at h11 region. All of them are repetitive homologous sequences differing in some features [crystal region exhibit crystalline aggregates in their spermatocytes [Stellate and crystal loci are normally silent; however, deficiencies in the crystal region lead to the de-repression of the Stellate sequences in the germline. At the molecular level, the loss of the crystal region results in the production of a testes-specific Stellate mRNA of 750 bases in length [Stellate mRNA, is the main component of crystalline aggregates [crystal–Stellate interaction among which are stingaub and scratchhsp83 [crystal-Stellate regulation occurs by the small-RNA-mediated pathway [crystal” piRNAs [crystal-Stellate regulation and the piRNA pathway. Indeed, the de-regulation of the crystal–Stellate interaction represents a simple readout to identify genes involved in the piRNA pathway in testes [crystal–Stellate interaction. The main features of several piRNA genes identified during the years are summarized in crystal-Stellate regulation are highlighted [crystal-Stellate modifiers are piRNA-related genes, although the opposite is not true. A crucial point regards Piwi that is not involved in the piRNA-mediated silencing of Stellate in the testes: nonetheless, it is fundamental to piRNA biogenesis in the ovaries. Since Piwi seems not to be required in the germline primary pathway, at least in the ovary [Stellate sequences mainly requires the germline primary pathway; this is also supported by the observation that the Stellate-related piRNAs share some signatures of the ping-pong pathway [Stellate sequences and of the transposons in testes (Stellate-related piRNA biogenesis in testes indicate that distinct pathways occur in the gonads of both sexes (the mechanism of primary piRNA production in the germline could be distinct from the piRNA production in the ovary) [The “eraction ,91. Stelfeatures ,93,94,95atocytes ,97. In an length ,99. In 1gregates . An earl3scratch ,99,101.  pathway ,102,103, pathway ,64,83,95n testes ,105. Mann testes ,106,107.hlighted ,127,128.he ovary ,82,129, n testes . Furthere ovary) ,87,89.Drosophila melanogaster [Drosophila mutants in the gonads and in the nervous system, along with the discovery of piRNAs in the nervous system of Drosophila and humans [Recently our group has demonstrated that dFmr1 plays a role in the piRNA pathway in testes and ovaries of nogaster . This stnogaster ,8. In add humans ,132,133,Stellate-made crystalline aggregates in spermatocytes of dFmr1∆50, dFmr1∆113 mutant males and in RNAi-dFmr1 males in which the function of the protein was selectively reduced in different cells of the gonads   , located at the N-terminal site to activate them ,121,138.dFmr1 mutant ovaries accompanied, as expected, by the reduction of fertility. Moreover, it is interesting to note that the expression patterns of dFmr1 and the piRNA-related protein Aubergine co-localize in specific territories of the ovary [Recently, dFmr1 has emerged as a member of piRNA-mediated pathways in ovaries ,139. Thehe ovary .Drosophila ovary is composed of 12 to 16 ovarioles. In each one, the most apical structure is the germarium, where stem cells are located. The developing egg chamber passes through the germarium and proceeds through its development, forming a linear chain. dFmr1 and Aubergine co-localize in the germarium, where they accumulate at the position of the stem cells. The two proteins also co-localize in the nuage of the nurse cells and, in the later stages, they overlap in the cytoplasm of the oocyte [The e oocyte . The rele oocyte . In parte oocyte . It is ke oocyte .bantam miRNA to control germline stem cells in the ovary of Drosophila [Drosophila, Ago1 is also required for the biological function of dFmr1 in neural development and synaptogenesis [In the ovary, dFmr1 is also involved in the microRNA-mediated pathway. The tight link between dFMR1 and the miRNA pathway has emerged from the interaction between the Fragile-X protein and the osophila . In humaosophila . In Drosogenesis .In the last part of this review we will examine the multiple roles and functions of the dFmr1 protein, following the threads of its genetic and biochemical/physical interactors in the gonads and in the germ granules representing a bridge from oocyte to embryos in the fly and other animals (mice and humans).dFmr1 has recently been associated with the piRNA pathway in the gonads of both sexes ,139 and The DEAD box RNA helicase Vasa is also considered a key component of the piRNA pathway; it is located at the nuage of ovaries and testes ,151,152 Another component of the germ granules at the pole plasm, where the pole cells will form, is the RNA binding protein Cup ,156,157.The P-bodies component, Me31B DEAD box helicase, represents a very intriguing link between Cup, Hsp83 and dFmr1 . Me31B cDrosophila dFmr1 and the G-quadruplets can be supported by the finding that one of the targets of dFmr1 in the nervous system is the protein Futsch, the homolog of the mammalian MAP1B, exhibiting a putative G-quadruplets in its 5′ end [Drosophila.An emerging function which has been demonstrated for the mammalian form of Fmr1 protein is related to its capacity to recognize the G-quadruplets, a specific structure in RNAs . It is is 5′ end ,144,153.Drosophila ovary [In addition to the cytoplasmic function of dFmr1, a nuclear function has been suggested due not only to the NLS and NES domains in the protein  1,24,16,1624,169la ovary . Zfrp8 pla ovary , and is la ovary ; it has la ovary ,175,176.Drosophila ovary, the loss of dFmr1 causes an incorrect localization of HP1 in the heterochromatin, suggesting a role for dFmr1 in chromatin state maintenance [The emerging role of dFmr1 in the nucleus is strongly supported by its connection with HP1. In the ntenance .Drosophila model for the discovery of new components of the piRNA pathway as well as for molecular studies of the piRNA process to be applied in mammals. dFmr1 is an RNA binding protein with multiple domains and multiple demonstrated functions. Some of these functions may be related to the different interactors in different tissues or in different subcellular populations, or both. In this review, following its interactors in the gonads, cells, and subcellular compartments, we have analyzed dFmr1’s roles in small RNA pathways: in regulating the localization of some nuage components, in shuttling between nucleus and cytoplasm, in chromatin remodeling, in binding the G-quadruplets and, finally, in potentially regulating mRNA translation in the ovary as well as at the synapses. All these studies indicate a conservation of the pathways involved in the regulation of transposons from the animal models to humans, and furthermore emphasize the importance of the It will be interesting, in the future, to further investigate the different roles of FMRP arising from its multiple interactors in different types of cells and tissues, in order to gain insights into its apparently different molecular roles that have a significant impact on human Fragile-X disease.


Andrias davidianus, by light microscopy, immunohistochemistry and transmission electron microscopy (TEM) methods. Toluidine blue staining showed TCs with one to two very thin and long telopodes (Tps) that were located in gastric lamina propria. Tps had characteristic structures, including podoms, podomers and dichotomous branching. Immunohistochemistry showed the existence of CD34+/PDGFRα+ TCs with moniliform Tps in stroma and were close to gastric glands and blood vessels. TEM micrographs also demonstrated the presence of TCs in interstitium between gastric glands. TCs/Tps were located in close proximity to gastric glands, blood vessels, endocrine cells and stem cells. In particular, Tps frequently surrounded stem cells. TCs and Tps, Tps and stem cells established close contacts. Moreover, the exosomes were also found near TCs/Tps. Our data confirmed the presence of TCs in gastric lamina propria of the amphibian, and suggested that TCs cooperate with resident stem cells to regulate endocrine cells and gastric glands regeneration and homeostasis.In this study, we attempt to identify gastric telocytes (TCs) of the Chinese giant salamander  Andrias davidianus (Amphibia: Caudata)A. davidianus and better understand TCs functions, in this study, we employ light microscopy, immunohistochemistry and transmission electron microscopy (TEM) methods, and attempt to identify TCs in the gastric lamina propria of A. davidianus.Telocytes (TCs) are a novel interstitial/stromal cell reported by Popescu’s team in 20101231147891012123491617181922122By toluidine blue staining, TCs with very thin and long Tps were observed in the interstitium between gastric glands . The cel+/PDGFRα+ TCs were located in interstitium between gastric glands  raised with simulated ecological breeding technology in their primary habitat were generously provided by an artificial breeding farm in mountain area, Jiangxi Province, China. The A. davidianus were euthanized on the ice, and then the stomachs were quickly excised and fixed by various fixative solutions for different methods, respectively. The sampling procedures and protocol were approved by the College of Animal Science and Technology, Jiangxi Agricultural University. All the measures are to alleviate animal’ s suffering.All procedures with the et al.A. davidianus were fixed in 10% neutral buffered formalin for 24 h at room temperature, and then the samples were washed with 0.01 M phosphate-buffered saline (PBS) at pH 7.4, dehydrated in series of graded ethanol, and embedded in paraffin. Sections (5 μm) were cut on a microtome . After deparaffinization, the sections were stained with toluidine blue solution (0.8% toluidine blue + 0.6% KMnO4) for 40 s. After washing in distilled water, the stained sections were dehydrated and mounted. Finally, they were examined and photographed by using a BM 2000 light microscopy with ScopeImage 9.0 (H3D) software .The light microscopy technique was conducted according to Zhang 2O2/methanol for 15 min. After washing the sections in PBS (0.01 M pH 7.4) for 3 × 5 min, they were pre-treated using heat mediated antigen retrieval with 0.01 M sodium citrate buffer (pH 6.0) for 15 min, and then washed in PBS for 3 × 5 min. All following steps were carried out in a moist chamber. The sections were incubated in normal goat serum at room temperature for 15 min. After discarding the goat serum, the sections were incubated in the anti-CD34 and anti-PDGFRα antibody  with diluted in PBS (1:200) at 4 °C overnight, respectively. After rinsing in PBS for 3 × 5 min, the sections were incubated with biotinylated anti-rabbit IgG at 37 °C for 30 min. Afterwards, they were detected using an HRP conjugated compact polymer system at 37 °C for 30 min. The 3,3-diaminobenzidin (DAB) was used as the chromogen. The colouration was terminated with PBS when positive cells were clearly visible. The sections were counterstained with haematoxylin and mounted. Finally, the sections were also photographed by using BM 2000 light microscopy.IHC was performed according to the protocol provided by the anti-CD34 and anti-PDGFRα antibody manufacturer . Briefly, after deparaffination, the sections (5 μm) were incubated in 3% Het al.et al.4  for 1 h, dehydrated in a concentration series of ethanol, infiltrated with propylene oxide–Araldite mixture and embedded in Araldite. The samples blocks were sectioned at 50 nm by using an ultramicrotome , and then the sections were mounted on cooper coated grids. Finally, these sections were stained with 1% uranyl acetate and Reynold’s lead citrate for 20 min. The stained sections were observed and photographed by a high resolution digital camera connected to the TEM .TEM was conducted according to methods of Yang The length of Tps was measured in the light microscopical and TEM images by using Image Pro-plus 7.0 software , respectively. Data were analyzed by using Excel software .How to cite this article: Zhang, H. et al. Telocytes in gastric lamina propria of the Chinese giant salamander, Andrias davidianus. Sci. Rep.6, 33554; doi: 10.1038/srep33554 (2016).


Working memory (WM) permits maintenance of information over brief delays and is an essential executive function. Unfortunately, WM is subject to age-related decline. Some evidence supports the use of transcranial direct current stimulation (tDCS) to improve visual WM. A gap in knowledge is an understanding of the mechanism characterizing these tDCS linked effects. To address this gap, we compared the effects of two tDCS montages designed on visual working memory (VWM) performance. The bifrontal montage was designed to stimulate the heightened bilateral frontal activity observed in aging adults. The unilateral frontoparietal montage was designed to stimulate activation patterns observed in young adults. Participants completed three sessions  of anodal tDCS . During stimulation, participants performed a visual long-term memory (LTM) control task and a visual WM task. There was no effect of tDCS on the LTM task. Participants receiving right unilateral tDCS showed a WM benefit. This pattern was most robust in older adults with low WM capacity. To address the concern that the key difference between the two tDCS montages could be tDCS over the posterior parietal cortex (PPC), we included new analyses from a previous study applying tDCS targeting the PPC paired with a recognition VWM task. No significant main effects were found. A subsequent experiment in young adults found no significant effect of either tDCS montage on either task. These data indicate that tDCS montage, age and WM capacity should be considered when designing tDCS protocols. We interpret these findings as suggestive that protocols designed to restore more youthful patterns of brain activity are superior to those that compensate for age-related changes. Visual working memory (VWM) allows us to integrate perception across interruptions such as eye movements. As we age, working memory (WM) performance, including VWM, shows decline  studies report that right posterior parietal cortex (PPC) activity reflects VWM load showing increases in BOLD signals until memory capacity plateaus . Furthermore, we were interested in determining whether these effects would generalize to young adults who already perform WM tasks at a superior level and rely on unilateral brain activation patterns. We completed a second experiment in young adults (Experiment 2).Here, we tested whether a montage designed to facilitate neural compensation or restoration was better at enhancing VWM performance in low and or high VWM capacity older adults. Regardless of the outcome, these data would be useful in tailoring tDCS protocols to maximize benefit. This answer may be important for enhancing the consistency of tDCS outcomes and for understanding the underlying mechanism of tDCS effects.α = 0.05 and estimating a modest effect size  task and a VWM task. The LTM task served as a control task because it was not expected to change as a function of tDCS so that it could confirm the limited nature of tDCS-linked benefits for each montage. The VWM task was the experimental task of greatest interest and followed our line of work showing tDCS-linked differential performance.Participants sequentially viewed 15 centrally located scenes  and made an indoor/outdoor (50%) judgment via button press response. LTM was tested in a recognition paradigm conducted after the VWM task. Participants indicated which of 30 scenes were old (50% old). There were four blocks of LTM task trials for a total of 120 recall trials; see Figures Participants viewed stimulus arrays containing three (50%) or six colored circles  drawn from a set of nine color patches. After a delay (1000 ms) one probe item appeared, and participants made a unspeeded old/new item-location recognition response via button press. In each block of trials participants completed 24 trials per set size and four total blocks for a total of 192 VWM trials; see Figure unilateral session, the anode was placed over right PFC (F6) and the cathode over right PPC (P6), or bilateral PFC-PFC , and sham . The same participants completed three counterbalanced tDCS sessions on different days separated by at least 24 h. Participants were also blinded to the nature of the stimulation. In the t(34) = −8.1, p < 0.00001). Due to the median split there were a total of 18 high/low VWM capacity participants. The resulting groups did not differ in terms of age , or MOCA  scores. To minimize between-subject variability we analyzed normalized difference indices derived from the accuracy data:High and low VWM capacity groups were formed using a median split on their Corsi Block Spatial Span scores  for each montage and for the VWM set size , and for the LTM task. These values were subjected to separate mixed analysis of variance (ANOVA) with the within-subjects factors of stimulation condition  and the between-subjects factor of VWM capacity .We modeled current flow to demonstrate the extent of current flow and to clarify the region of field intensity across the whole brain using HD-Explore™/tDCS-Explore software ; see Figure t-test. The normalized LTM DI from unilateral and bilateral showed no significant difference between unilateral (Mean (M) = −0.002, SD = 0.04) and bilateral stimulation  conditions (t(35) = 0.84, p = 0.4). There was no effect of tDCS montage on LTM task performance.To address the specificity of any tDCS-montage effects, we examined LTM performance using a paired sample F = 10.04, p = 0.003, partial η2 = 0.23), such that unilateral stimulation provided greater benefit to VWM than bilateral stimulation. There was also a borderline significant main effect of VWM capacity  = 3.6, p = 0.06, partial η2 = 0.096) showing that the low VWM capacity participants received the greatest benefit. Accordingly, there was a significant interaction of montage × VWM capacity  = 10.41, p = 0.003, partial η2 = 0.23). This interaction can be explained such that the low VWM capacity participants benefited from unilateral stimulation, whereas the high VWM capacity participants showed little effect of tDCS montage or set size; see Table Fs < 1, p > 0.2 for set size, set size × VWM capacity, and montage × set size × VWM capacity, montage × set size).To test whether the tDCS montages differentially modulated VWM performance, DIs were subjected to mixed effects ANOVA with the within-subjects factors of montage  and set size 3, 6), and the between subject factor of VWM capacity ; see Figure , 6, and F,34 = 10.Experiment 1A showed that older adults with low VWM capacity benefited from the unilateral frontoparietal tDCS montage. One possible interpretation of these data is that the unilateral montage serves to restore age-related reductions in frontoparietal networks underlying VWM. Whereas the bifrontal montage did not successfully benefit VWM performance, the role that VWM capacity played in this process remains significant but difficult to predict. Additionally, because the difference between the two montages an alternative interpretation was that the effect was driven by tDCS targeting the right PPC site. In other words, potentially, the benefit was exclusively due to current reaching the PPC, rather than unilateral or bifrontal stimulation. Finally, important to note that behavioral performance did not reach ceiling (>90% accuracy) or floor (below chance) indicating that our task was neither too easy or too difficult; see Table To address the concern that tDCS over PPC targets served as the key difference between the two tDCS montages, we include a new analysis from a previously published study , PPC 64.72(5.72)), education , PPC 16.94(3.57)), and MMSE score , PPC 28.33(1.5)).In the full study, there were 72 neurotypical self-reporting right-handed older adults who scored a 25 or higher on the Mini-Mental Status Examination (MMSE). Participants were randomly assigned to one of four groups . Here, we select sham and PPC groups alone for new analysis. Thus, 36 older adults , divided into two groups similar in age  and the cathode over the right contralateral cheek (CC). tDCS  was delivered through two 5 × 7 cmt(8) = −6.2, p < 0.00001), PPC  = −6.2, p < 0.00001) the groups do not different in terms of age: high WMC (t(8) = 0.05, p < 0.96), low WMC (t(8) = 0, p < 1) or MMSE: high WMC (t(8) = 0.6, p < 0.56), low WMC (t(8) = 0.42, p < = 0.67). Groups included nine participants each.The high and low VWM capacity groups were formed by median split based on OSpan scores: sham  = 0.56, p = 0.46, partial η2 = 0.01), or VWM capacity  = 0.08, p = 0.78, partial η2 = 0.002) on VWM accuracy. No interactions approached significance . The results of the ANOVA show that anodal tDCS to PPC did not alter VWM performance.A two-way ANOVA examined the effect of tDCS  and VWM capacity  on VWM accuracy; see Figure These data provide some insight as to whether the tDCS-linked VWM benefit observed in Experiment 1A could be entirely attributed to right PPC stimulation. Despite several paradigmatic differences between Experiments 1A and 1B, the data provide some confirmation that one session of right PPC stimulation is not sufficient to bolster VWM performance in these sorts of VWM tests probed by recognition. Important to note that behavioral performance did not reach ceiling (>90% accuracy) or floor (below chance) in this task as well; see Table Experiment 1A revealed a benefit of unilateral tDCS compared to bilateral frontal tDCS exclusively in the low VWM capacity group. Because younger adults show greater hemispheric lateralization than older adults, this raised the question of whether we might improve VWM performance in younger adults when applying a unilateral montage, or whether in this population heightening frontal activity via bilateral frontal tDCS. In essence, this was the inverse of our predictions for the healthy older adults. This experiment also extended our previous findings showing improved VWM in high VWM capacity participants after anodal or cathodal tDCS targeting the right posterior parietal lobe, whereas those with low VWM capacity showed impairment in both cases  participated. The experimental procedures remained consistent with Experiment 1A except that the younger adult group did not perform the MOCA.t(35) = 6.4, p < 0.00001), the resulting groups were not different in terms of age (p = 0.68) there were a total of 18 participants in each group.The high and low VWM capacity groups were formed by conducting a median split based on their Corsi Block Spatial Span scores  and bilateral stimulation  = 1.6, p = 0.1). The tDCS montage did not significantly impact LTM task performance; see Figure Difference indices were no different for either condition: unilateral  = 0, p = 1, partial η2 = 0), set size  = 0.30, p = 0.58, partial η2 = 0.01), or capacity:  = 2.97, p = 0.09). No interactions were noted . There was a non-significant numerical trend showing improved VWM performance in the high WM capacity group after either tDCS montage , VWM capacity , and set size 3, 6) revealed no significant main effects of stimulation .adults  = 9.94, p = 0.002, partial η2 = 0.12). Neither the main effect of montage or the interaction approached significance  and between-subject factors of age  confirmed superior performance in the young F = 5.63, p = 0.02, partial η2 = 0.08) reflecting the VWM benefit of unilateral tDCS. No other main effects approached significance . There was also an intriguing interaction of montage × age  = 5.62, p = 0.02, partial η2 = 0.07) such that older adults garnered greater benefit from the unilateral montage compared to the younger adults; see Tables F = 6.56, p = 0.01, partial η2 = 0.09) revealing greater benefits in high VWM capacity young adults and low VWM capacity older adults. Of greatest interest was the significant three-way interaction of montage × VWM capacity × age  = 9.12, p = 0.004, partial η2 = 0.12) again reflecting the greater benefit from the unilateral montage provided a VWM performance benefit to older adults with low VWM capacity (SS 3: 0.041 (SD: 0.073), SS 6: 0.043 (0.05)), and to younger adults with high VWM capacity (SS 3: 0.023 (SD: 0.06), SS 6: 0.01 (0.04)). No other interactions reached significance  = 3.3, p = 0.07, montage × set size × age: F = 2.34, p = 0.14).A 2  × 2  × 2  × 2  mixed effects ANOVA using the DI data revealed a significant main effect of montage . An additional, related challenge is that some individual differences predict an individual’s behavioral response to tDCS, but which ones are consequential and why remains unclear. Finally, a challenge for tDCS application has been the enormous parameter space and little clear optimization, and few experimental approaches relying on theoretically grounded alternative hypotheses.Given these unknowns, we initiated the current experiments to test whether healthy older adults would receive greater VWM benefit from a frontoparietal tDCS montage that was hypothesized to restore a more lateralized pattern of VWM-related brain activity, or from a bifrontal montage hypothesized to support compensatory bilateral frontal activity. The data showed that the healthy older adults’ performance on the VWM change detection task improved when the unilateral tDCS montage was applied to restore a more youthful pattern of brain activations. However, this VWM performance benefit was limited to the older adults with lower WM capacity. The high WM capacity group of older adults showed no notable change in VWM or LTM performance for either tDCS montage. Next, we reanalyzed existing data to ensure that it was right unilateral stimulation that was driving our significant main-effect of stimulation. The data showed that stimulation to PPC in older adults showed no benefit of stimulation following a single session of tDCS when compared to a control group. This supports our findings that restoration of a more youthful pattern of brain activity is beneficial to older adults with WM capacity. We also tested high and low WM capacity younger adults to see if there were differential benefits associated with either tDCS montage. Here, the logic was that reinforcing their more lateralized pattern of activity might support VWM performance, or alternatively, by supporting greater bilateral frontal activity VWM performance might improve. The data showed that there was a VWM benefit associated with the unilateral montage but in the high WM capacity group. Instead of clearly supporting a uniform tDCS protocol to enhance WM across participants, or across participants with a particular WM capacity, these data highlight the difficulty of anticipating how individual differences affect responsiveness to tDCS. The data confirm that age and WM capacity are important factors that should be considered when designing tDCS-linked experiments or therapeutic interventions.A serious challenge in interpreting several critical meta-analyses of tDCS-linked effects on WM (Horvath et al., either anodal or cathodal tDCS targeting the posterior parietal improved VWM performance in older adults with more education (Berryhill and Jones, Several years ago, we were surprised to find that Finally, what is striking about these significant individual differences in single-session per condition designs is that all older adults in a longitudinal design targeting either the PFC, the PPC, or alternating between both sites, showed WM benefits (Jones et al., We acknowledge that to be more generalizable we would want to include a wider variety of WM tasks. A limitation of this work is that the present study involved testing only a single VWM task. We also tested VWM alone and implemented a right-lateralized unilateral tDCS protocol. To understand whether verbal WM performance responds similarly a left-lateralized tDCS experiment is needed. Despite these limitations, our observation that the behavioral consequences of tDCS depend on who is doing the participating and where the stimulation is targeting. These data do suggest that the strategy involved in conducting the task will be important to specify in going forward. We also acknowledge that our hypotheses about effects are derived from advances in current modeling which has validated success in predicting the regions affected by tDCS (Bikson et al., HA, FG and MEB conceived and designed the experiment. HA and FG collected behavioral-tDCS data and analyzed behavioral data. HA and MEB contributed to writing and revising the manuscript. KTJ and JAS conceived, designed, collected behavioral-tDCS data for Experiment 1B.The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest.


EIF2B1–5, encoding five subunits of eukaryotic translation initiation factor 2B (eIF2B). Although the defects are in the housekeeping genes, glial cells are selectively involved in VWM. Several studies have suggested that astrocytes are central in the pathogenesis of VWM. However, the exact pathomechanism remains unknown, and no model for VWM induced pluripotent stem cells (iPSCs) has been established.Vanishing white matter disease (VWM) is an inherited leukoencephalopathy in children attributed to mutations in Fibroblasts from two VWM children were reprogrammed into iPSCs by using a virus‐free nonintegrating episomal vector system. Control and VWM iPSCs were sequentially differentiated into neural stem cells (NSCs) and then into neural cells, including neurons, oligodendrocytes (OLs), and astrocytes.Vanishing white matter disease iPSC‐derived NSCs can normally differentiate into neurons, oligodendrocytes precursor cells (OPCs), and oligodendrocytes in vitro. By contrast, VWM astrocytes were dysmorphic and characterized by shorter processes. Moreover, δ‐GFAP and αB‐Crystalline were significantly increased in addition to increased early and total apoptosis.The results provided further evidence supporting the central role of astrocytic dysfunction. The establishment of VWM‐specific iPSC models provides a platform for exploring the pathogenesis of VWM and future drug screening. Two age‐matched control iPSCs (C1 and C2) were obtained from the Stem Cell Research Center at Peking University. The HDFs were cultured in a standard medium containing high‐glucose DMEM supplemented with 10% fetal bovine serum (FBS). iPSCs were maintained in Essential 8 medium  on Matrigel . iPSCs were passaged every 3‐5 days by EDTA (Life Technology).2.25) were counted and resuspended in the Nucleofector solution supplied in the Amaxa Nucleofector kit . The episomal plasmids were added to the cell suspensions at 10 μg each per reaction and were cotransfected using the program U‐023 on the Amaxa Nucleofector device. The cells were then transferred to a Matrigel‐coated 60‐mm culture dish and cultured in the fibroblast medium for 3 days, and then changed to N2B27 medium supplemented with bFGF , Y‐27632 , CHIR99021 , PD0325901 (Selleck), hLIF (Invitrogen), and A‐83‐01 (Selleck). On day 12, the medium was changed to E8. By 20‐30 days post‐transfection, clones were picked and cultured in E8 medium.Yamanaka episomal plasmids used in experiments, including pCXLE‐hOCT3/4‐SHP53 (#27‐007), PCXLE‐hSK (#27078), pCXLE‐hUL (#27080), and pCXLE‐EGFAP (#27082) . HDFs (5 × 102.37 cells/mL) were suspended in PBS and injected subcutaneously into the posterior limbs of 4‐week‐old NOD/SCID mice. Two months after injection, the teratomas were dissected and fixed in 4% paraformaldehyde. The paraffin‐embedded tissue was sliced and stained with hematoxylin and eosin.Chromosomal G‐band analysis was performed on VWM iPSCs with more than 10 passages. Alkaline phosphatase (ALP) staining was performed by using an ALP kit (Invitrogen). Teratoma assay was performed to test the in vivo differentiation potential of iPSCs. The undifferentiated iPSCs . cDNA was synthesized using a reverse transcription kit . Quantitative PCR was performed using SYBR Green Real‐time PCR Master Mix (Promega). The relative expression levels were normalized to those of GAPDH based on the delta‐Ct method. The forward and reverse primers for real‐time PCR are as follows: GAPDH ; Total GFAP; α‐GFAP ; δ‐GFAP.2.5Cells were fixed with 4% paraformaldehyde. The primary antibodies included OCT4 , SOX2 , SSEA4 , MBP , GFAP , S100β , Nestin , Neurofilament H , and βIII‐tubulin , PDGFRα, NG2 , αB‐Crystalline . Secondary antibodies 488/568 goat antimouse/rabbit IgG (Invitrogen) were used and diluted at 1:500. The cell nuclei were counterstained with Hoechst33342 (Invitrogen). The labeled cells were imaged with a confocal microscope .2.6Vanishing white matter disease and control iPSCs more than 10 passages were differentiated into NSCs and then sequentially differentiated into neural cells, including neurons, oligodendrocytes (OLs), and astrocytes. Markers in different differentiation stages were detected. The fluorescence density was quantitatively detected by the following method: 8‐10 fields were randomly selected, including at least five areas in each field, then the mean fluorescence density in each area was calculated.2.6.1Induced pluripotent stem cells colonies were digested with EDTA when the iPSCs reached 70%‐80% confluence and then cultured in an E8 medium for a day. For 2‐7 days, the cells were cultured in a neural induction medium (Stemcell), and NSCs were formed and cultured in an NSC medium . Immunofluorescence assay was used to detect the markers Nestin and SOX2.2.6.2Neural stem cells were digested into single cells and then plated onto a Matrigel‐coated culture dish and cultured in a neuronal medium (bFGF removed from the NSC medium). By 7 days, the markers βIII‐tubulin and Neurofilament H were detected.2.6.3Neural stem cells were dissociated into single cells using Accutase and cultured in an astrocyte differentiation medium (Stemcell), and the cells were passaged every 4 days, both Nestin and CD44, indicative of immature astrocytes, were detected by immunofluorescence assay. By 12 days, the cells were digested and cultured in an astrocyte maturation medium (Stemcell). After at least one passages, the markers of GFAP and S100β, which are indicative of mature astrocytes, were detected by immunofluorescence assay. The number of processes and the length of the longest process from the margin of the nuclei were measured by calculating 10 randomly selected fields (cell number >5).2.6.44/cm2. The next day, the culture medium was changed to OL precursor cell (OPC) medium . After 7 days, the markers of NG2 and PDGFRα, which are indicative of OPCs, were detected by immunofluorescence assay. To differentiate the OPCs into OLs, the OPCs were dissociated into single cells and plate‐cultured in a wall of a six‐well plate coated with Matrigel at a density of 5 × 104/cm2. The next day, the culture medium was changed to an OL medium .Neural stem cells were dissociated into single cells and transferred to a wall of a six‐well plate coated with Matrigel at a density of 5 × 102.7The cells were digested with trypsin without EDTA. According to the instructions of the apoptosis kit , 100 μL of binding buffer solution was added to each sample, mixed with 2 μL of Annexin V and 2 μL of PI, and light reaction was avoided for 15 minutes at room temperature. Apoptosis was detected by flow cytometry  within 1 hour and analyzed with FlowJo software .2.8For detecting total GFAP protein expression in astrocytes, total protein was extracted by RIPA buffer. Antibody to GFAP  was used in routine Western blot analysis. The expression of GAPDH  was used as a loading control.2.9P < 0.05 was considered statistically significant.Statistical analysis was performed with SPSS 20.0 . The ANOVA analysis was adopted to determine the statistical significance in the following assays: the number of processes and the length of the longest astrocytic process, apoptosis detection, fluorescence density, 2.10This study was approved by the clinical research ethics committee of the Peking University First Hospital, and informed consent forms were signed by the parents of the patients.33.1EIF2B5 and EIF2B3 compound heterozygous mutations, respectively, were established using a nonintegrating episomal vector system. Both patients were early childhood onset VWM, the developmental milestone before disease onset was normal. The first patient (VWM1) was a male, whose initial symptom was motor deterioration triggered by head trauma at 4 years old. At the last follow‐up in 2018, he was 16 years old and was bedridden. The second patient (VWM2) was a female, who was characterized by motor regression at the age of 3 and died at 12 years old. The brain MRI showed typical features of rarefaction of cerebral white matter , c.1157G>A ; whereas the genotype of VWM2 was EIF2B3: c.140G>A (p. Gly47Glu), c.1037T>C (p. Ile346Thr) , which are 3.2Forearm dermal tissue was obtained from the two VWM patients at the age of 10 and 11, respectively. To generate integration‐free iPSCs, the Yamanaka episomal plasmids were electrotransfected into the HDFs of the two VWM patients using an Amaxa Nucleofector system. 25‐30 days after electrotransfection, clones were picked and propagated under feeder‐free conditions. iPSCs clones exhibited compact and flat appearance, similar to ESCs. In addition, iPSCs stained positive for alkaline phosphatase (ALP) staining and expressed typical pluripotent markers, SSEA4, and NANOG  were induced to differentiate into NSCs by using neural induction medium. After two passages, both control and VWM iPSCs expressed Nestin and SOX2. Nestin was localized in the cytoplasm, whereas SOX2 was in the nuclei Figure a. In add5 Figure b.3.4P > 0.05, whereas those of Neurofilament H were 814.8, 744.5, 841.3 and 831.5, respectively, P > 0.05   Figure c.P > 0.05) . CD44 was localized in the membrane, and the mean fluorescence densities of CD44 in the C1, C2, VWM1, and VWM2 cells were 856.1, 840.9, 838.1, and 843.4, respectively (P > 0.05) , and those of S100β were 897.9, 923.8, 918.9, and 889.6 (P > 0.05) . The numbers of processes between the control and VWM astrocytes did not demonstrate significant difference, P > 0.1 , mitochondrial dysfunction, and glial maturation dysfunction.4.2The typical brain MRI of VWM patients shows that the cerebral white matter is diffusely rarefied, whereas the cortex is relatively well preserved.4.3In the central nervous system (CNS), astrocytes account for the largest number of cells and play a central role in the maintenance of homeostasis, the response to injury, and the pathogenesis of disease. Astrocytes participate in a series of complex pathological processes, such as reactive gliosis, antioxidant, and immune regulation.EIF2B5 mutation; they found that few GFAP+astrocytes were present and astrocytic induction was severely compromised, whereas normal OLs can be cultured. Detailed VWM pathological examination has revealed meager reactive astrogliosis, dysmorphic astrocytes, and increased expression of delta isoform GFAP (δ‐GFAP) and heat shock protein αB‐crystalline.Several studies have suggested that astrocytes are central in the pathogenesis of VWM. Dietrich et alIn our study, we found that VWM iPSC‐derived NSCs can normally differentiate into OPCs, and OLs in vitro. Whereas, VWM iPSC‐derived astrocytes were dysmorphic, expressed a significant increased δ‐GFAP and αB‐Crystalline, and showed increased early and total apoptosis as well, which indicating the astrocytic dysfunction. Dysmorphic astrocytes overexpressed δ‐GFAP, suggesting that the intermediate fiber network of VWM astrocytes was affected, resulting in abnormal morphology and meager astrogliosis.In addition to the pathological changes in astrocytes in cerebral white matter, astrocytes in other regions can also be involved. Both in VWM patients and mouse model of VWM, Bergmann glia in the cerebellum and Müller cells in the retina are also affected. The Bergmann glia in the cerebellum shows mislocalization to the molecular layer, and the involvement of Müller glia was manifested as retinal dysplasia. Retrospective evaluation of patients’ electroretinographic data confirmed that retinopathy is also a sign of VWM patients.4.4There must be some differences between iPSC differentiation in vitro and neural differentiation in vivo. Moreover, although iPSCs carry VWM mutations, new variants, and epigenetic changes may occur during the processes of iPSCs reprogramming and differentiation.5In this study, two VWM iPSC models were established using a virus‐free nonintegrating episomal vector system for the first time. The results suggested that there was no difference in the in vitro differentiation of control and VWM iPSCs into NSCs, neurons, and OLs. Whereas, VWM astrocytes exhibited abnormal morphology, increased expression of δ‐GFAP and αB‐Crystalline, and increased early and total apoptosis, further supporting that astrocytes may play a central role in the pathogenesis of VWM. In addition, the established VWM‐specific iPSCs models provide a platform for further study on the pathogenesis and future drug screening.The authors declare no conflict of interest.


Making workers aware of the risks they incur is key to implementing hearing preservation initiatives, but some occupations present intractable challenges. Gary Humphreys reports. People’s disregard for the fragility of their ears is a source of constant surprise to Dr Shelly Chadha. “People put on sunglasses to protect their eyes from ultraviolet light, but you rarely see people wearing ear plugs,” she says. “In fact, the tendency is to wear ear phones to play amplified music, often at harmful levels.”Chadha, who heads up the World Health Organization’s (WHO) work on deafness prevention, believes that one of the reasons for this discrepancy is a failure to appreciate just how fragile ears are.“The threshold at which damage starts to occur to hearing is just 85 decibels,” she says. “That level can easily be reached in a noisy restaurant or in a street where there is heavy traffic or construction.”Noise-induced hearing loss usually begins with declining sensitivity to sound at high frequencies and typically shows up on audiograms as a dip or ‘notch’ at 4000 Hertz. “If noise exposure continues, this notch gradually deepens and spreads as the hair cells in the cochlea and the cells that support them deteriorate,” Chadha explains. “Once the damage is done there is no going back.”Notwithstanding the challenges epidemiologists face in characterising the disease burden imposed by excessive noise, it is widely assumed that most noise-induced hearing loss occurs in the workplace. “Noise is present to some degree in almost all workplaces,” says Dr Ivan Ivanov, team lead, Global Occupational and Workplace Health at WHO headquarters. “It can be caused by a sudden sound, such as the bang of a rivet gun or by continuous exposure to loud sounds over an extended period, such as the noise generated by an assembly line.”The main approaches to preventing noise-induced hearing loss are: to reduce the noise generated in the workplace, for example by investing in quieter machinery or moving machines away from where employees are normally located (referred to as engineering controls), to organize work in a way that limits worker exposure to noise (referred to as administrative controls) and to protect people’s ears by ensuring they wear earplugs or other protective devices.“The priority is to eliminate the noise hazard,” says explains Dr Manal Azzi, an occupational safety and health specialist at the International Labour Organization (ILO). “If that is not possible, efforts should focus on engineering and administrative controls to mitigate the harm for workers, such as limiting the time they are exposed to the noise hazard. Relying on personal protective equipment should be a last resort.”In recent years, workplace hearing preservation initiatives have been supported by technology, including personal sound exposure meters. The size of a bottle cap, these dosemeters are usually worn on the shoulder and measure the noise levels workers are exposed to throughout their shift, allowing employers to establish which areas may present a noise hazard and/or which employees are most exposed.“Once the damage is done there is no going back.”Shelly Chadha.Wall-mounted sound monitoring devices, featuring luminous panels with icons corresponding to different sound levels, are also used to raise awareness among workers regarding their exposure and thus encourage noise reduction or the use of hearing protection. For example, such devices are being used by the car manufacturer Audi at worker training facilities in Ingolstadt and Neckarsulm, Germany and have been used in a critical care unit in Preston, England.Unfortunately, many occupations preclude the elimination of noise sources, and have a limited scope for shielding workers from noise. Construction workers are among those with the highest levels of noise-induced hearing loss.In most high-income countries hearing conservation measures are backed by regulation, that derives from ILO’s Working Environment  Convention of 1977 (updated in 2001) and includes the United States of America’s (USA) Occupational Noise Exposure Standard and the European Union’s (EU) Physical Agents Noise Directive.In low-income countries the regulatory environment is less supportive of hearing conservation efforts, while the informal sector is largely unregulated. “Most informal sector workers lack social protection, rights at work and decent working conditions,” Ivanov says, “and that includes working conditions safe for people’s hearing.”One occupation often criticized in this regard is ship recycling. Previously located in Taiwan, China and the Republic of Korea, the bulk of ship recycling is now carried out in Bangladesh, China, India, Pakistan and Turkey, for the most part without effective occupational safety regulation.One of the largest shipbreaking facilities in the world is in the coastal town of Alang, in Bhavnagar district, Gujarat, India. Alang is home to some 120 ship-recycling companies, employing more than 40 000 workers. Located in the nearby city Ahmedabad, Joydeep Majumder, a workplace noise and audiometry expert, working at the National Institute of Occupational Health, is familiar with conditions in Alang and identifies two key concerns. The first is the way work is organized, with workers exposed for prolonged periods to harmful noise. “Oxy-fuel torch cutters generally work in the same area all day and can be exposed to levels of sound exceeding 140 decibels for prolonged periods,” Majumder says. He has petitioned ship recycling companies to allow workers to change task or location on the work site, but with limited success.The second concern is the lack of personal protection or rather the use of personal protection by workers. “Workers do not want to use ear plugs or ear muffs because they cannot hear each other and because of the humidity. They are sweating heavily and the ear plugs are uncomfortable. They also fear getting ear infections.”“Relying on personal protective equipment should be a last resort.”Manal AzziFor Majumder improving awareness of the risks to which ship recyclers are exposed is key to improving the uptake and use of personal protection. “The majority of these workers are not even aware that they have a problem,” he says. “They typically come from rural areas and are surrounded by other hearing-impaired people and they are all shouting, and they do not realize what is happening until they go back to their families.” Majumder has been trying to increase awareness and encourage personal protection use by conducting regular training sessions on site with colleagues.WHO’s Chadha agrees that raising awareness is vital. “The earlier workers become aware of the damage being done to their hearing, the more likely they are to protect themselves,” she says, emphasizing the importance of regular audiological assessment for early identification of onset of hearing issues. International convention for the safe and environmentally sound recycling of ships, referred to as the Hong Kong Convention, which includes provisions for hearing protection.According to Rohit Agarwal, managing partner of Guideship Consulting Services, a company offering consultancy services to ship recycling companies in Alang, working conditions are improving, as companies strive to comply with the International Maritime Organization’s “Around 80 of the Alang yards have achieved Hong Kong Convention compliance certificates and more are undergoing audits for certification,” he says, arguing that the pressure for change is coming from responsible ship owners. “They not only sell their vessels to Hong Kong Convention-compliant yards, but appoint third-party supervisors to monitor activities and ensure standards are maintained.”  Ship recyclers are also competing for European Union flagged vessels, which since 2018, can only be recycled in facilities that are compliant with the above-mentioned EU directive. To date, none of the Indian recyclers has been included.For ILO’s Azzi, the conditions faced by India’s ship recyclers typify challenges faced by many of the estimated two billion people now employed in informal work, most of them in emerging and developing countries. “Improving workers’ hearing health and their health generally, depends on establishing safe and healthy working environments,” Azzi says. “Achieving that will require a transition to the formal economy.”


MicroRNAs (miRNAs) act a significant role in multiple biological processes and their associations with the development of all kinds of complex diseases are much close. In the research area of biology, medicine, and bioinformatics, prediction of potential miRNA-disease associations (MDAs) on the base of a variety of heterogeneous biological datasets in a short time is an important subject. Therefore, we proposed the model of Composite Network based inference for MiRNA-Disease Association prediction (CNMDA) through applying random walk to a multi-level composite network constructed by heterogeneous dataset of disease, long noncoding RNA (lncRNA) and miRNA. The results showed that CNMDA achieved an AUC of 0.8547 in leave-one-out cross validation and an AUC of 0.8533+/−0.0009 in 5-fold cross validation. In addition, we employed CNMDA to infer novel miRNAs for kidney neoplasms, breast neoplasms and lung neoplasms on the base of HMDD v2.0. Also, we employed the approach for lung neoplasms on the base of HMDD v1.0 and for breast neoplasms that have no known related miRNAs. It was found that CNMDA could be seen as an applicable tool for potential MDAs prediction. Unlike the miRNAs, long noncoding RNAs (lncRNAs) are a sort of heterogeneous ncRNAs with about 200 nucleotides and usually show less sequence conservation. Accumulating evidence indicates that miRNAs are participated in a wide variety of life process of cells, such as proliferation2, development3, aging4, viral infection5, metabolism6 and so on7. It is no surprise that miRNAs are closely related to a number of clinically important diseases9. For example, miR-335 and miR-126 were proved to be metastasis suppressor miRNAs in human breast cancer10. In addition, previous study also confirmed that the differential expression of miR-21, -31, -143 and -145 is closely participate in clinic pathologic features of colorectal cancer11. Therefore, identification of disease-related miRNAs would be beneficial for disease diagnosis, treatment, and prevention12. Currently, unlike traditional time-consuming biological experiments, adopting validation to the predicted miRNA-disease associations (MDAs) obtained from calculation models could reduce a lot of time and cost. Therefore, it is very significant to propose effective calculation models to infer potential MDAs17.MicroRNAs (miRNAs) is a kind of short noncoding RNA (ncRNA) molecules with about 22 nucleotides in length which can regulate complementary messenger RNAset al.18 developed a computational approach based on matrix completion, in which the adjacency matrix constructed from known MDAs was updated to gain final association scores of each miRNA-disease pair. Considering various types of known MDAs, Chen et al.19 constructed an restricted boltzmann machine (RBM) model to further predict four kinds of MDAs.According to the idea that miRNAs with similar functions are usually relevant to similar diseases and the reverse is also true. some researchers built elaborate computational models for the identification of potential MDAs on the basis of known MDAs in databases only. For example, Li et al.20 developed an effective method via combining all those information to construct a heterogeneous graph and then further inferred MDAs with the consideration of paths between miRNA nodes and disease nodes. Besides, this method could also be implemented to predict for new diseases (miRNAs). Through integrating the distribution information of k most similar neighbors per miRNA and the corresponding functional similarity between the miRNA and its neighbors, Xuan et al.21 proposed a reliable computational approach to infer novel MDAs. However, HDMP cannot predict disease-related miRNAs for new diseases. After computing miRNAs functional similarity (MFS), Xuan et al.22 proposed a prediction model via implementing random walk on constructed miRNA functional similarity network in which they assigned larger transition weights to marked nodes. At last, probability association scores of each disease-miRNA pair would be obtained and ranked. A calculation model was further built by Chen et al.17 in which miRNA’s k-nearest-neighbors (KNNs) and disease’s KNNs were respectively searched and then these KNNs would be ranked according to support vector machine. After that, they finally got all potential MDAs with weighted voting. Under the framework of semi-supervised learning, a novel model23 was presented for MDAs prediction via combining the optimal solutions in the miRNA space and disease space. Recently, Chen et al.24 proposed another prediction model through calculating within-score and between-score for both miRNAs and diseases which were then combined to obtain the final MDA scores.Based on the information of known MDAs and the corresponding similarity information of diseases and miRNAs, Chen et al.25 presented a prediction model on the basis of the constructed integrated network. By connecting miRNAs to diseases with the proteins as a bridge between them, a calculation model was employed by Mork et al.26 through using a scoring scheme, which can greatly increase the model’s efficiency. Furthermore, Shi et al.27 implemented random walk on a built protein similarity network to identify MDAs.Also, researchers put forward some other calculation approaches via considering relevant genes or proteins as a bridge to predict novel MDAs. For example, using a discrete probability distribution of hypergeometric, Jiang et al.28 by the analyzed of random walk with restart (RWR). It is worth noting that RWR is a very effective model for MDAs prediction. By adopting RWR, a novel model named Composite Network based inference for MiRNA-Disease Association prediction (CNMDA) was presented in the light of a multi-level network which was built by combination of Gaussian interaction profile kernel similarity (GIPKS) for lncRNAs, integrated similarity for miRNAs (ISMs) and diseases (ISDs), known MDAs, lncRNA-disease associations (LDAs) and miRNA-lncRNA interactions (MLIs). In addition, leave-one-out cross validation (LOOCV) and 5-fold cross validation were adopted in this paper to assess CNMDA’s effectiveness. It could be seen that the AUCs of LOOCV and 5-fold cross validation were respectively 0.8547 and 0.8533+/−0.0009. As for case studies, CNMDA was carried out on kidney neoplasms (KN), breast neoplasms (BN) and lung neoplasms (LN) to infer its associated miRNAs based on HMDD v2.029. Also according to HMDD v2.0, we further infer novel miRNAs for BN after hiding its known associated miRNAs. At last, we carried out the case studies based on HMDD v1.030 to infer LN-related miRNAs. Based on the above results, the effectiveness of CNMDA for MDAs prediction was validated.By combining the known MDAs network and MFS network, a new calculating method was studied by Chen 29 and then made comparison between CNMDA and four other classical computational models: RLSMDA23, HDMP21, WBSMDA24 and RKNNMDA17  would be utilized to validate the top 50 identified outcomes. In the second case study, we respectively inferred BN-associated miRNAs and BN-associated miRNAs after removing all known BN-associated miRNAs in HMDD v2.0. In the third kind of case studies, CNMDA was adopted to predict for LN according to associations in HMDD v1.0 and v2.0, respectively.31. If kidney tumors are detected and treated early and localized in the kidney, Patients would have a good disease-specific survival rate. Otherwise, patients have only an 18% two-year survival rate when they present with terminal disease32. With recent researches and studies, about two hundred and fifty thousand renal tumor patients are newly diagnosed annually, and KN’ morbidity and mortality continue to increase33. Many miRNAs related to KN have been found based on a large number of biological experiments. For example, in renal cell carcinoma (RCC), up regulation of miR-21 is related to kidney cancer that with lower survival rate34. Through targeting MMP-9 in RCC, miRNA-133b can suppress cell proliferation, migration and invasion35. Finally, we implemented CNMDA for potential KN-related miRNA prediction. It was found that 8 of the first 10 and 37 of the first 50 miRNAs were verified 40. In order to test the stability of CNMDA, we employed CNMDA based on the associations in HMDD v2.0 and HMDD v1.030, respectively. It was found that 20 and 28 of first 50 associated miRNAs for LN have been verified, respectively . From the evaluation results, it can be seen that the accuracy of our prediction model was superior in the comparison with other four models.As overwhelming evidences expounded that miRNAs are participated in all sorts of diseases. The development of new calculation approaches for predicting MDAs in a short time is important to further experimental validation. Accordingly, it is now possible to confirmed novel MDAs using biological experiments with low time and cost. Existing models are usually proposed based on four different calculation mechanisms42, LDAs43 and drug-target interaction44. Furthermore, CNMDA could identify novel diseases that have no known associated miRNAs. At last, the implementation of CNMDA only needs positive samples as training data. Since there is no known negative sample information, the forecasting precision of CNMDA is more convincing. However, some limitations also exist in the computation model of CNMDA. For example, the number of experimentally determined MDAs, LDAs and MLI is insufficient. For the number of known MDAs, only 5430 known MDAs were collected. The more the known MDAs, the higher forecasting precision the model. Importantly, the current forecasting precision still needs to be improved according to the evaluation of LOOCV.The main merits for the effective performance of CNMDA are as follows: Through taking advantage of multi-source information based on reliable database, it is no surprise that the integration strategy of CNMDA could predict potential MDAs effectively. Secondly, in comparison of local network information, RWR is an iterative process based on global network for the MDAs prediction. The attractive properties of global network information have been proved in the identification for potential disease-gene associations, MDAs29. Through constructing a adjacency matrix Wdm to indicate the 5430 known MDAs, we made use of variables nm and nd to express the total amount of miRNAs and diseases in the known MDAs dataset, respectively.Experimentally confirmed MDAs used in this paper were come from high-quality database45. After removing excess LDAs whose diseases don’t arise in the 5430 known MDAs mentioned above, we would acquire 250 known LDAs. Likewise, we built an adjacency matrix Wdl to indicate the 250 known LDAs. Variable nl refer to the number of lncRNAs in the 250 known LDAs.The known LDAs was from the LncRNADisease46. In the same way, we need to delete excess MLIs whose miRNAs and lncRNAs do not exist in the 5430 known MDAs and 250 known LDAs. At last, 9088 known MLIs were gotten and an adjacency matrix Wml was used to refer to the 9088 MLIs.The known MLIs was from starBase v2.0http://www.cuilab.cn/files/images/cuilab/misim.zip47. We used FS to indicate the score of MFS between miRNA m(i) and miRNA m(j).The scores of MFS were obtained from 48 on the basis of Directed Acyclic Graph (DAG)49, which can be picked up according to MeSH descriptor of Category C. In the DAG = , E(D)) for disease D, all nodes are linked together from father to son using a straight line. The nodes of D and its elder can be collected into T(D) and E(D) referring to all the straight lines from father to son. Therefore, contribution of disease d in DAG(D) to the semantic value of disease D can be put forward.D to its own semantic value is 1. The semantic value of disease D could be put forward.We put forward DSS1d(i) and d(j) can be described.At last, DSS1 between 48, due to the fact that a more specific disease d appearing in less DAGs would contribute more to the semantic value of disease D. Accordingly, the contribution made by d for the semantic value of D can be described byIn the DSS2d(i) and d(j) can be defined as follows:DSS2 between disease d(u), we used IP(d(u)) to refer to row vectors of line u in Wdm on the basis of known MDA. Through watching whether d(u) is related to each miRNA, we computed GIPKS for diseases d(u) and d(v)50.For disease m(i) and m(j) can be constructed.Similarly, GIPKS for miRNA l(p) and l(q), GIPKS between them can be constructed.For lncRNA d(u) and d(v)24.We have taken into account combining GIPKS for diseases, DSS1 and DSS2 to compute ISD between diseases m(i) and m(j) can be put forward by the integration of GIPK for miRNA and MFS24.Similarly, the ISM between miRNAs et al.51). In our introduced model, we used T refer to the transposition of matrix.Aiming at the prediction of potential MDAs, a computing method of CNMDA was stated. Carrying out RWR on a multi-level composite network that built by integration of ISM, ISD, GIPKS for lncRNA, known MDAs, LDAs and MLIs, final association scores of novel MDAs would be obtained  denote the weight of ISD network, ISM network and the network of GIPKS for lncRNAs, respectively. The corresponding u0, v0, w0 are the original probabilities of these three-similarity networks respectively. Here, u0 is calculated through assigning equal probability to all nodes in LDAs with a total to 1. Similarly, v0, w0 can be calculated.Global information based on the multi-level network would be captured through RWR algorithm. At each steps, seed nodes move to their immediate neighbors with a probability W. M represents the transition probability from i to j. In the network of GIPKS for lncRNAs, the transition probability from lncRNA i(li) to lncRNA j(lj) was put forward.Meanwhile, the transition matrix i(di) to disease j(dj) was put forward.Similarly, in the ISD network, the transition probability from disease In the ISM network, the transition probability from miRNA i(li) to disease j(dj) was put forward.In the LDAs network, the transition probability from lncRNA i(li) to miRNA j(mj) was put forward.In the MLIs network, transition probability from lncRNA In the LDAs network, the transition probability from disease In the MDAs network, the transition probability from disease In the MLIs network, the transition probability from miRNA Pt and −6). Then, the candidate miRNAs can be ranked according to In the MDAs network, the transition probability from miRNA 51, which used RWR on the same multi-level composite network in their study. Therefore, we set the parameter x, y, z, α, β to By incorporating MLIs and LDA into MDAs prediction, RWR was put forward on a constructed multi-level network to infer novel MDAs. In the network, because initial MLIs, LDAs and MDAs have more credibility, they all as weights in the RWR equations. Obviously, the one interaction and two associations play an equally important part in the network to disseminate information of miRNAs, diseases and lncRNAs for the novel MDAs prediction. In this study, we chose the same parameter as the one in previous literatureSupplementary materialSupplementary Table 1Supplementary Table 2Supplementary Table 3Supplementary Table 4Supplementary Table 5Supplementary Table 6


An increase in naturally-occurring porphyrins has been described in the blood of subjects bearing different kinds of tumors, including colorectal, and this is probably related to a systemic alteration of heme metabolism induced by tumor cells. The aim of our study was to develop an artificial neural network (ANN) classifier for early detection of colorectal adenocarcinoma based on plasma porphyrin accumulation and risk factors.(ROC) curve to assess performance in distinguishing colorectal adenocarcinoma patients and controls. A liquid chromatography-high resolution mass spectrometry (LC-HRMS) analytical method was employed to identify the agents responsible for native fluorescence.We measured the endogenous fluorescence of blood plasma in 100 colorectal adenocarcinoma patients and 112 controls using a conventional spectrofluorometer. Height, weight, personal and family medical history, use of alcohol, red meat, vegetables and tobacco were all recorded. An ANN model was built up from demographic data and from the integral of the fluorescence emission peak in the range 610–650 nm. We used the Receiver Operating Characteristic p < 0.0001) and was weakly correlated with the TNM staging . LC-HRMS measurements showed that the agents responsible for the fluorescence emission were mainly protoporphyrin-IX (PpIX) and coproporphyrin-I (CpI). The overall accuracy of our ANN model was 88% (87% sensitivity and 90% specificity) with an area under the ROC curve of 0.83.The fluorescence analysis indicated that the integral of the fluorescence emission peak in the range 610–650 nm was significantly higher in colorectal adenocarcinoma patients than controls (These results confirm that tumor cells accumulate a diagnostic level of endogenous porphyrin compounds and suggest that plasma porphyrin concentrations, indirectly measured through fluorescence analysis, may be useful, together with risk factors, as a clinical decision support tool for the early detection of colorectal adenocarcinoma. Our future efforts will be aimed at examining how plasma porphyrin accumulation correlates with survival and response to therapy. About 90% of people whose colorectal cancer (CRC) is diagnosed before it has spread to nearby lymph nodes or organs  survive more than five years after diagnosis. However, only 14% of those whose cancer has spread to distant parts of the body (distant stage) survive five years , 3, the Currently the fecal immunochemical test (FIT) is considered the screening test of choice for CRC: it shows greater sensitivity than the guaiac-based fecal occult blood test (gFOBT) , 5, is mThere are several biomolecules which, when excited at suitable wavelengths, give fluorescence emission over a wide spectral range. As a pathological condition develops in tissue, changes occur in biochemical, physicochemical and histological properties at the cellular and tissue levels and the fluorescence emission spectrum may show changes, facilitating the distinction between normal and malignant tissue. This finding has been already applied in the early detection of breast, cervix, colorectal and oral cancer –13.In previous works , 15, we In this pilot study we used the integral of the fluorescence emission peak in the range 610–650 nm, hereafter referred to as IF-INT, and the demographic data of the subjects enrolled to train an artificial neural network (ANN) multiparametric test for colorectal adenocarcinoma. The primary objective was to verify the performance of the classifier for distinguishing patients with colorectal adenocarcinoma from control subjects. The second endpoint was to identify the agents responsible for the fluorescence signal by liquid chromatography-high resolution mass spectrometry (LC-HRMS).Between January 2013 and December 2014, we consecutively recruited all colorectal adenocarcinoma patients who accessed the Colon-Rectal Surgery Unit of the Fondazione IRCCS Istituto Nazionale Tumori in Milan, Italy, and who agreed to participate in the study. Patients were eligible if they were more than 18 years old, had never had other tumors, had no concomitant non-tumor gastrointestinal diseases such as Crohn’s disease and diverticulitis, had not undergone chemotherapy in the six months prior to admission and had a histopathologic diagnosis of colorectal adenocarcinoma. A blood sample was taken before surgery from all patients meeting the selection criteria. In the same period, we recruited controls among the patients who entered the Endoscopy Unit of our Institute to undergo colonoscopy for family history, following a positive outcome of FIT or gFoBT or for a bleeding episode that required immediate diagnostic assessment. Subjects were eligible if they were more than 18 years old, had never had cancer, did not have concomitant gastrointestinal diseases such as Crohn’s disease and diverticulitis, and colonoscopy confirmed the absence of colorectal carcinoma, adenomas or inherited syndromes. A blood sample was taken before colonoscopy from all the patients meeting the selection criteria.All the subjects enrolled gave written informed consent to participate and completed a questionnaire to record information on height and weight, alcohol intake , red meat  and vegetables , smoking status  and family history of CRC .The Ethics Committee of our Institute approved this study protocol before subjects were enrolled.p value < 0.05 was taken to indicate statistical significance.Blood samples were collected in lithium-heparin tubes, centrifuged and the supernatant was collected. All supernatants were stored at − 20 °C until analysis. The fluorescence analysis method has been described in detail previously . BrieflyThe predictive performance of IF-INT was assessed from the area under the receiver operating characteristic curve (AUROC). Sensitivity, specificity, negative predictive value (NPV) and positive predictive value (PPV) were also calculated.Artificial neural networks are ideal for modeling non-linear relationships between a set of predictors or input variables and one or more responses or output variables. The action of an ANN is defined by the neurons of each layer, which are the basic computational units of the network, and by the connections between the layers, with their weights. Among all the possible network architectures, the feed-forward neural network with back-propagation training has been widely adopted for realistic nonlinear multiple regression in different medical fields . In a feIn order to develop a classifier to distinguish colorectal adenocarcinoma patients from controls, we built up a feed-forward ANN model using MATLAB software . Seven variables were considered as inputs for the network: IF-INT, body mass index (BMI), calculated by dividing the weight in kilograms by the square of the height in meters, alcohol consumption, red meat intake, vegetables intake, smoking status and family history of CRC. A three-layer structure was used with only one hidden layer; during network instruction, the number of hidden neurons was varied between 1 and 6. The output of our model was between 0 and 1.The first phase of model development is the training procedure on a defined set of input variables with known output data. The overall population was randomly divided into three subsets, training, validation and test sets, and the back-propagation algorithm with the early stopping procedure was applied; the leave-one-out cross-validation tool was adopted in order to avoid overfitting the data . MultiplAfter optimizing the ANN model, the relative importance (RI%) of the input variables was assessed through the most squares method  to evaluSeveral studies suggest that tumor cells are able to produce porphyrins naturally or after administering their precursor , 21–23 av/v) were added to 400 μL of plasma; after vortexing and centrifugation at 17000 relative centrifugal force (RCF-g) for 20 min at 15 °C, the supernatant was transferred into a polypropylene tube. The organic layer was evaporated in a stream of nitrogen at 40 °C. The dry residue of plasma samples was reconstituted with 100 μL water: aceto- nitrile:acetic acid 2:1:1 v/v/v and 15 μL were injected into the LC-HRMS system.In detail, 1000 μL of ethyl acetate:acetic acid  equipped with an XBridge BEH300-C18 3.5 μm, 150 × 2.1 mm column . A binary mobile phase and gradient elution were used at a flow rate of 350 μL/min. Mobile phases were: A, HAn Orbitrap Elite high-resolution mass spectrometer  was used as detector, working in electro-spray ionization mode in positive polarity: spray voltage was 4.5 kV; sheath and auxiliary gas (nitrogen) pressure were 40 and 15 a.u. respectively; the vaporizer temperature was 320 °C; the capillary temperature was 350 °C; s-lens was 69 V. M/Z values were acquired in full scan (FS) mode (mass range 250–1300 Da); resolving power was set to 120.000 full width at half maximum (FWHM) to permit exact mass extraction of molecular ions from the FS spectra; chromatographic RTs were used to ensure reliable compound identification.Five analytes were selected that are involved in heme biosynthesis and have characteristic fluorescence emission in the range 610–650 nm: protoporphyrin-IX (PpIX), coproporphyrin-I (CpI), iron protoporphyrin IX (FePpIX), biliverdin (Bv) and protoporphyrin IX dimethyl ester (dmPpIX) . These analytes, each at a concentration of 1 mg/ml, were used to prepare a standard solution. Twenty plasma samples with and 20 without evidence of native fluorescence, and the porphyrin standard solution, were processed with the analytical method.From January 2013 to December 2014, 135 patients who accessed the colorectal surgery unit of our Institute were consecutively enrolled. Fifteen were excluded for missing information; 12 were non-eligible because they had received chemotherapy in the six months prior to blood sampling, five patients because their blood sample was hemolyzed and therefore not analyzable; three were excluded after surgery as they were found not to have adenocarcinoma. The colorectal adenocarcinoma patients were classified postoperatively according to the TNM-UICC classification based on clinical and pathological findings. Seven patients were stage 0, 31 stage I, 29 stage II, 25 stage III, and 8 stage IV. In the same period, 157 patients who accessed the endoscopy unit of our Institute were recruited. Thirty-one were excluded as the colonoscopy and the histological exam confirmed the presence of an adenoma, familial adenomatous polyposis or Crohn’s disease; 14 patients were excluded for missing information.2), normal (18.50–24.99), overweight (25.00–29.99) and obese (≥30.00) [. Distributions of BMI, smoking, alcohol and meat consumption by sex indicated a healthier lifestyle for women than men. In addition, differences in BMI and alcohol and meat consumption between colorectal adenocarcinoma patients and control groups were more marked for men than women. Among men, colorectal adenocarcinoma patients were less frequently normal weight and more frequently overweight, had higher alcohol and red meat intake. As regards smoking, there seemed to be no marked differences between patients and controls, of either sex. No significant differences were observed in vegetable intake between colorectal adenocarcinoma patients and controls, or between men and women. Finally, a family history of CRC was definitely more frequent among controls than cases. The controls in our study were in fact pre-selected subjects at risk for whom a colonoscopy had been prescribed, while in the patients the disease had actually manifested itself.The main demographic characteristics of the study population are summarized in Table (≥30.00) . ColorecP < 0.0001. The Spearman’s rho correlation coefficient between IF-INT and TNM staging was 0.224 (P = 0.011), suggesting that there is a weak positive linear correlation between IF-INT and disease severity. With the arbitrary cut-off of 125.00 a.u., the overall accuracy of IF-INT in correctly distinguishing between colorectal adenocarcinoma patients and controls was 84%, with 85% sensitivity and 79% specificity. The AUROC was 0.786.As found in our previous studies , 15, the2 of 0.987. Table The optimized ANN architecture was achieved using five hidden neurons; the best validation was reached after 157 iterations, with MSE 0.079 and a coefficient of determination RTable Figure In tumor cells the synthesis of PpIX is highly activated and ferrochelatase mRNA expression is down-regulated so PpIX tends to accumulate specifically in tumor tissues , 28. TreIn this study we examined the potential utility of the plasma concentrations of intrinsic porphyrin compounds in the early diagnosis of colorectal adenocarcinoma. We found significantly higher endogenous porphyrin concentrations in the plasma of colorectal adenocarcinoma patients than control subjects; this was indirectly evident from analysis of the native fluorescence spectrum and directly confirmed by LC-HRMS analysis. Fluorescence analysis is a very sensitive technique, easy and quick to implement but it cannot precisely determine the agent responsible for the fluorimetric signal, since different compounds may have the same fluorescence spectrum. LC-HRMS is much more laborious and expensive but does determine the agents responsible for the fluorescence signal by calculating the mass of the substances in the sample.In the presented method, the analytical descriptor selected to distinguish between colorectal adenocarcinoma patients and control subjects is the integral of the fluorescence emission peak between 610 nm and 650 nm, acquired with a conventional fluorimeter following blue light irradiation. According to our LC-HRMS analysis, the agents responsible for plasma fluorescence emission in the selected wavelength range were mainly protoporphyrin-IX and coproporphyrin-I. Both these compounds have fluorescence emission in the range 610–650 nm when excited at 405 nm, with maximum emission wavelengths at respectively 622 and 632 nm . InvestiAbout 75% of CRC patients have sporadic forms of the disease. The remaining 25% have a family history of colorectal cancer, adenomatous polyps or inherited syndromes such as familial adenomatous polyposis (FAP) and Lynch syndrome, suggesting a contribution of inherited genes, shared environmental factors, or some combination of these , 4, 32. We found the overall accuracy of IF-INT for colorectal adenocarcinoma detection was 84%, with 85% sensitivity and 79% specificity, while the overall accuracy of the ANN model (IF-INT plus risk factors) was 88%, with 87% sensitivity and 90% specificity. Although the relative importance of each individual risk factor is significantly lower than that of porphyrin concentration, the contribution of risk factors is not irrelevant in the diagnostic performance of the ANN model and could hopefully be increased considering other risk factors currently unverifiable on the our study population. Accuracy, sensitivity and specificity of our ANN model are comparable to the performances of FITs for colorectal cancer detection, as reported in a meta-analysis performed by Lee et al.  that sugThe results reported in our paper confirm the presence of diagnostic levels of endogenous porphyrin compounds in the blood plasma of colorectal adenocarcinoma patients and suggest that the measurement of plasma porphyrins concentration may be applied, together with risk factors, as a clinical decision support tool for the early detection of colorectal adenocarcinoma. Further investigations of selected CRC patients are under way to assess IF-INT’s utility as a marker that correlates with survival and response to therapy.


Asynchronous automated telehealth-based hearing screening and diagnostic testing can be used within the rural school context to identify and confirm hearing loss.The aims of the study were to evaluate the efficacy of an asynchronous telehealth-based service delivery model using automated technology for screening and diagnostic testing as well as to describe the prevalence, type and degree of hearing loss.A comparative within-subject design was used. Frequency distributions, sensitivity, specificity scores as well as the positive and negative predictive values were calculated. Testing was conducted in a non-sound-treated classroom within a school environment on 73 participants (146 ears). The sensitivity and specificity rates were 65.2% and 100%, respectively. Diagnostic accuracy was 91.7% and the negative predictive values (NPV) and positive predictive values (PPV) were 93.8% and 100%, respectively.Results revealed that 23 ears of 20 participants (16%) presented with hearing loss. Twelve per cent of ears presented with unilateral hearing impairment and 4% with bilateral hearing loss. Mild hearing loss was identified as most prevalent (8% of ears). Eight ears obtained false-negative results and presented with mild low- to mid-frequency hearing loss. The sensitivity rate for the study was low and was attributed to plausible reasons relating to test accuracy, child-related variables and mild low-frequency sensory-neural hearing loss.The study demonstrates that asynchronous telehealth-based automated hearing testing within the school context can be used to facilitate early identification of hearing loss; however, further research and development into protocol formulation, ongoing device monitoring and facilitator training is required to improve test sensitivity and ensure accuracy of results. Children comprise 32 million of the 360 million people globally presenting with disabling hearing loss, and it is estimated that almost 9% of hearing-impaired children are 15 years and younger  conducted a synchronous tele-hearing screening service in a school. Remote computing software was used so that the audiologist could remotely operate the audiometer whilst the facilitator set up and instructed the participant at another school. TeamViewer was used to provide videoconferencing and synchronous services between both sites for screening to be conducted. Thirty-one children participated in the study. All participants were screened using telehealth and conventional methods of screening with no statistically significant differences between methods. In the studies conducted by both Swanepoel et al. and Saleth et al., only manual audiometry was utilised which is different from the context of the present study, which aims to evaluate the use of automated audiometry within the natural school environment.A telehealth model using synchronous audiometry was used to conduct hearing screening on 143 children in a study by Skarżyński et al. . HearingThe purpose of the present study was to build on the work conducted in automated audiometry by evaluating the efficacy of an asynchronous model using automated audiometry to conduct screening and diagnostic testing. This was considered necessary because most of the studies reviewed have either conducted synchronous testing or used manual audiometry as opposed to automated audiometry.The aims of the study were to evaluate the efficacy of an asynchronous telehealth-based service delivery model using automated technology for screening and diagnostic testing in a rural school in South Africa as well as to describe the prevalence, type and degree of hearing loss found in the study sample.A comparative within-subject design was used. A within-subject design ensures that every participant receives the test that is under investigation and serves as his or her own control. This reduces the amount of error that may arise when comparing variables between participants  was invited to participate and were given invitation letters, along with consent forms, information documents and letters of assent to take home to their parents. Only those learners who returned a signed consent form were included in the study.The entire population of learners at the school (= 1.99). Both genders were adequately represented in the study sample with 60% girls and 40% boys.Participants were from Grades 1 to 7 and ranged in age from 6 to 12 years with a mean age of 8.9 . The KUDUwave 5000 was connected to an HP ProBook laptop with a Windows 10 operating system. The KUDUwave provides ‘Ambidome’ technology, which has built-in soundproofing, replacing the soundproof booth  and was computer literate. The facilitator was trained by the primary investigator (SG) who had been trained by a KUDUwave representative. Facilitator training took place over 2 days. Day one consisted of an overview of the hearing screening protocol and orientation to the KUDUwave and the digital Firefly otoscope. Aspects including proper handling and setting up of the equipment, patient set-up, patient information entry and troubleshooting were addressed. Day two comprised a practical component where the assistant was required to conduct screening of 10 children under the observation of the primary investigator. The primary investigator was also present for the first day of screening to ensure that no problems were experienced by the facilitator. The assistant was also given a copy of the KUDUwave manual  to ensure that the noise levels were kept below 50 dBSPL. There were only a few occasions when the noise levels reached an average of 60 dBA after the three readings and testing was interrupted during these times.Participants were conditioned for both screening and diagnostic testing before the assessment began. They had the opportunity to ask questions, to listen to practice tones and were orientated to the response button. Instructions were given in a simple manner in the first language of the participant.The screening protocol as outlined by SASLHA  was adheEach child was screened bilaterally with the left ear tested first, using automated screening methods at frequencies from 500 Hz to 4000 Hz. The pass criterion was set at 25 dBHL  after the participant was set up and instructed by the facilitator. Automated audiometry was used to conduct the diagnostic phase of testing. All participants were given a rest period of an hour or longer between screening and diagnostic testing. Six grades of hearing loss, from slight to profound, were classified according to Clark . The cliAll data were saved to a password-protected folder with only the researchers having access. Data were entered and verified by both researchers. All forms and audiograms were stored in a locked cabinet after data entry was complete.Frequency distributions were calculated for all screening and diagnostic tests. Sensitivity (%) and specificity (%) scores as well as the positive and negative predictive values (PPV/NPV) were calculated using two-by-two contingency tables. Sensitivity reflects the probability of the automated test being positive when the hearing loss is truly present. Specificity indicated the probability of the automated test being negative when the hearing loss was truly absent. PPV represents the percentage of participants with a positive result who actually have hearing loss, whilst NPV reflects the percentage of participants with a negative test outcome who truly do not present with hearing loss.Ethics approval was obtained from the Biomedical Research and Ethics Committee of the University of KwaZulu-Natal (certification number: BE288/15). Ethics approval was also obtained from the Department of Basic Education within the North West province. Participants received information letters, consent forms and letters of assent. Participants were informed of their right to withdraw at any stage of the study. Participants who required management were appropriately referred, and all participants received a copy of their results to take home to their parents. Data were entered onto a password-protected computer, and names were replaced with research numbers to ensure anonymity and confidentiality.A total of 146 ears were assessed. For those participants who were rescreened at one or more frequencies, only the rescreen results were used for analysis.Based on otoscopic findings, four ears (3%) required referral for cerumen management, three for partial impaction of wax and one for complete occlusion. They were referred for cerumen removal then rescreened. These participants also underwent diagnostic audiometric testing.In all, 15 ears (10.3%) of 13 participants failed the automated screening test. A total of 16 participants were rescreened after obtaining a fail result on the initial screen and 13 participants obtained a fail result on the rescreen, whilst 3 participants passed the rescreen and were given an overall pass result. For diagnostic testing, 23 ears (15.7%) of 20 children were identified as having a hearing deficit. Eight ears that passed the screening test result failed the diagnostic test. Therefore, there were eight false-negative screening tests but no false-positive tests. The sensitivity, specificity, PPV, NPV and diagnostic accuracy of the automated test are shown in All hearing losses identified were sensory-neural in nature. Mild hearing loss was identified in 11 ears (8%), moderate in 7 (5%), severe in 3 (2%) and profound in 2 ears (1%). Seventeen ears (12%) presented with unilateral hearing impairment and six ears of three participants (4%) had bilateral hearing impairment.The eight false-negative screening test results were further analysed . Three eThis study showed the results of a combination of asynchronous telehealth and automated testing methods to deliver both screening and diagnostic hearing services in a rural school context. Diagnostic testing performed at the school overcomes the risk of loss to follow-up for diagnostic testing that occurs in conventional screening services. The present study had a high specificity (100%) but a lower sensitivity (65.2%). This is similar to the findings of Cardoso et al.  where thIt is understood no screening measure is likely to identify all true positives and true negatives Stein, , becauseThe false-negative tests in this study are of concern. Whilst the authors could not find a definitive explanation, possible reasons for the false-negative tests could relate to the device, the testing procedure or child-related variables. Distractibility of some of the participants during diagnostic testing was a concern, especially the younger ones (6-year olds). Because of this, they may have not been focused during diagnostic testing, therefore failing the test. The diagnostic and screening tests used the same device, so tone generation and volume should have been consistent. The KUDUwave device has been validated for diagnostic testing in several studies  of false-negative results (Hyde, Riko, & Malizia, Careful protocol consideration is imperative when conducting a hearing evaluation programme. Although not investigated in the study, the extension of the response time on the automated software provided more time for the learner to respond. This is especially necessary when evaluating children because of their uncertainty and fluctuating attention levels. Screening protocols should also carefully consider their criteria in terms of screening intensity levels. The majority of the screening protocols recommend that screening be conducted at 20 dBHL SASLHA, ; howeverThe study found that children presented with primarily unilateral mild hearing impairment. This finding was consistent with other studies, which indicate that this type of hearing loss is prevalent in the school-aged population (Butler, This study confirmed that a facilitator can be trained to perform video-otoscopy and pure-tone screening using an automated audiometric device in a school during teaching hours. The results can be transmitted to an audiologist for review at a later time. Diagnostic testing can also be performed in the school using the same device. For this study diagnostic testing was undertaken by an audiologist on site, but it can also be performed remotely by an audiologist with the facilitator setting up the patient and being supervised in real-time over the Internet. Automated audiometry because of its predetermined protocol also allows for an adequately trained facilitator to execute the test under the remote observation of the audiologist. The results could then be forwarded to an audiologist for later review and management decisions. For the developing world, task shifting and enabling a facilitator to perform both screening and diagnostic testing on site in a school would be the most efficient use of scarce resources. Other similar studies also document successful outcomes when using a trained facilitator in an underserved context (Biagio, Swanepoel, Adeyemo, Hall, & Vinck, The study investigated the outcomes of a telehealth-based audiology service model using automated audiometric testing to screen hearing and confirm hearing loss in children at a rural South African school. The findings revealed that automated audiometry used within an asynchronous model can identify hearing loss in school-aged children; however, the issue of the false-negative rate obtained in this study was of concern. It is therefore recommended that ongoing evaluation occurs for automated devices to improve their reliability and validity particularly in relation to child-related factors. When testing children, it is important to ensure that they are attentive and alert to avoid effects of distractibility on test results. Counterbalancing testing order especially with children is important so as to avoid a learning effect for listening, and this was a limitation of the study probably contributing to the high false-negative rate. These factors support the increasing need for practice guidelines to be developed for tele-audiology to ensure standardised practice. Future research should focus on developing other automated audiology tests and implementing these within the school context so that a comprehensive test battery of automated audiology tests can be provided within a telehealth model.