Anopheles gambiae, and discovered that an infection upregulates multiple components of the immune deficiency (IMD) and c-Jun N-terminal kinase (JNK) pathways in the heart with periostial haemocytes. This upregulation is greater in the heart with periostial haemocytes than in the circulating haemocytes or the entire abdomen. RNA interference-based knockdown then showed that the IMD and JNK pathways drive periostial haemocyte aggregation and alter phagocytosis and melanization on the heart, thereby demonstrating that these pathways regulate the functional integration between the immune and circulatory systems. Understanding how insects fight infection lays the foundation for novel strategies that could protect beneficial insects and harm detrimental ones.The immune and circulatory systems of animals are functionally integrated. In mammals, the spleen and lymph nodes filter and destroy microbes circulating in the blood and lymph, respectively. In insects, immune cells that surround the heart valves (ostia), called periostial haemocytes, destroy pathogens in the areas of the body that experience the swiftest haemolymph (blood) flow. An infection recruits additional periostial haemocytes, amplifying heart-associated immune responses. Although the structural mechanics of periostial haemocyte aggregation have been defined, the genetic factors that regulate this process remain less understood. Here, we conducted RNA sequencing in the African malaria mosquito,  These gambiae –9, heart gambiae –12. Furt gambiae .Escherichia coli but not Staphylococcus aureus [In mosquitoes, pattern recognition receptors in the thioester-containing protein family, together with adhesion and phagocytosis factors in the Nimrod protein family, positively regulate immune responses on the heart ,15. Howes aureus . The Jaks aureus ,23, and s aureus ,25.A. gambiae. Using an unbiased RNA sequencing (RNAseq) approach that was followed by empirical testing with RNA interference (RNAi), we uncovered that the IMD and JNK pathways drive periostial haemocyte aggregation and immune responses on the heart, thereby regulating the functional integration between the immune and circulatory systems of mosquitoes.The goal of this study was to identify genes and regulatory pathways that drive periostial haemocyte aggregation in . 2. 2.1E. coli or Gram(+) S. aureus . Three tissues were isolated at 4 h after treatment: (i) the heart containing periostial haemocytes, (ii) the haemolymph containing circulating haemocytes and (iii) the entire abdomen. Mosquitoes were assayed at 4 h after treatment because the number of periostial haemocytes approximately doubles within the first hour of infection, and plateaus by 4 h after infection [E. coli and S. aureus infection, respectively .To identify genes that may drive periostial haemocyte aggregation, we undertook an unbiased RNAseq approach where we sequenced the transcriptome of tissues from mosquitoes that were naive, injured or infected with Gram(–) GFP-nfection . In thesp < 0.05) in the heart and periostial haemocytes of mosquitoes infected with GFP-E. coli and S. aureus, respectively (d). Because periostial haemocyte aggregation is a dynamic immune response that occurs during any bacterial infection, we hypothesized that its core molecular drivers must be preferentially upregulated in periostial haemocytes following both E. coli and S. aureus infection. We found that 55 genes met this overlap criterion . Comparing the heart and periostial haemocyte RNAseq data to RNAseq data from (i) circulating haemocytes and (ii) the entire abdomen then revealed that infection upregulates more genes in the heart with periostial haemocytes than in the circulating haemocytes or the abdomen .RNAseq revealed that, relative to the heart and periostial haemocytes of naive and injured mosquitoes, 73 and 132 genes are upregulated more than fourfold (ectively d. Becausf), and strikingly, six are part of IMD and JNK pathway: the peptidoglycan recognition protein PGRP-LA [IAP2 [rel2 [cec3 [def1 [JNK3 [PGRP-LA and IAP2, and downstream elements from both branches of the pathway, with the classical IMD cascade represented by rel2, cec3 and def1, and the bifurcation into the JNK pathway represented by JNK3 [eater [TEP1, which is involved in phagocytosis and periostial haemocyte aggregation [Drosophila inhibit the IMD pathway [TGase3—negatively regulates periostial haemocyte aggregation and has infection-dependent effects on the heart rate [draper [TEP1 [CLIPB17, DDC, SRPN16, CLIPB11, PGRP-LB and upd3a—do not participate in a common pathway.We then scrutinized the reported or predicted function of the 55 candidate genes. Twelve have immune function f, and st PGRP-LA , the inhLA [IAP2 , the IMDP2 [rel2 ,29, the c3 [def1 ,31 and tf1 [JNK3 ,33. Mult by JNK3 ,34,35 . Five of these genes were shared between the heart with periostial haemocytes and the circulating haemocytes, but none of the 43 genes have a classical role in immunity or carry a function suggestive of involvement in haemocyte aggregation. Therefore, based on the RNAseq analyses, we hypothesized that the main driver of periostial haemocyte aggregation is the IMD and JNK pathway.Because the downregulation of genes could also impact haemocyte aggregation, we assessed the genes that were downregulated after both . 2.2rel2, and its negative regulator, caspar, and achieved RNAi-based silencing of 43% and 37%, respectively, compared to the dsR)bla. Moreover, because Tep1 is (i) activated by the Rel2 arm of the IMD pathway [rel2 and caspar RNAi mosquitoes. Silencing of rel2 did not alter TEP1 mRNA abundance but silencing of caspar increased it. Because cec3 and def1 are also activated by Rel2 [rel2 decreased cec3 and def1 mRNA abundance whereas silencing of caspar increased them . Overall, these data are consistent with the role that the Rel2 arm of the IMD pathway plays in regulating TEP1, cec3 and def1 expression.To determine whether the IMD pathway regulates periostial haemocyte aggregation, we synthesized double-stranded RNA (dsRNA) to target the IMD cascade transcription factor,  pathway ,45 and bla(Ap control mosquitoes (figures R)blabla. At 24 h post-infection, the effect of rel2 RNAi on periostial haemocyte aggregation was diminished. When we instead knocked down caspar, the number of periostial haemocytes increased by 39% in uninfected mosquitoes, and infection for 4 and 24 h increased the number of periostial haemocytes by 30% and 19%, respectively, relative to similarly treated dsR)bla. Knockdown of rel2 or caspar did not alter the number of non-periostial sessile haemocytes in uninfected mosquitoes or mosquitoes infected for 4 h. However, at 24 h following infection, caspar RNAi decreased non-periostial sessile haemocytes by 22%. These results demonstrate that the IMD pathway regulates periostial haemocyte aggregation while having a minimal effect on the rest of the sessile haemocytes.To determine whether the IMD pathway specifically affects periostial haemocytes and not sessile haemocytes in general, we counted the number of sessile haemocytes outside of the periostial regions in the tergum of abdominal segments 4 and 5 in the same mosquitoes examined for periostial haemocytes c. Knockd-E. coli fluorescence pixel area in the periostial regions of infected mosquitoes . At 4 h following infection, the GFP-E. coli pixel area was similar regardless of dsRNA treatment. At 24 h after infection, this area decreased in dsR)bla. Melanin was absent in uninfected mosquitoes and mosquitoes that were infected for 4 h. However, at 24 h following infection, rel2 RNAi increased melanin deposits at the periostial regions whereas caspar RNAi had no effect.Periostial haemocytes phagocytose bacteria, leading to their accumulation at the periostial regions ,6. To debacteria ,6, and oria [DDC ,47—was uE. coli and melanin in rel2 RNAi mosquitoes at 24 h after infection could be due to either (i) enhanced phagocytosis by periostial haemocytes or (ii) higher bacterial proliferation in the haemocoel, which places increased pressure on the phagocytosis response. To differentiate between these two scenarios, we measured the systemic GFP-E. coli infection intensity and observed that, at 4 h after infection, the bacterial intensity was similar for all dsRNA treatments, but at 24 h, knockdown of rel2 resulted in a higher infection intensity than treatment with dscaspar or dsR)bla(Ap (c). This suggests that rel2 is essential for proper bacterial killing in the haemocoel, and therefore, knocking it down increases infection intensity in a manner that leads to increased phagocytosis in the periostial regions. However, because silencing rel2 and caspar had opposite effects on periostial haemocyte aggregation at 4 h, a time when dsRNA treatment does not impact infection intensity, we conclude that the IMD pathway is a positive regulator of periostial haemocyte aggregation.The increased accumulation of GFP-bla(ApR) c. This sDrosophila haemocytes [f). Therefore, we tested the involvement of PGRP-LA in periostial haemocyte aggregation. PGRP-LA has three splice forms, so to knock it down, we synthesized dsPGRP-LA-RARB to target the RA and RB splice forms, and dsPGRP-LA-RC to target the RC splice form. Using these dsRNAs, we achieved RNAi-based silencing that ranged from 56% to 77%, compared to the dsR)bla. PGRP-LA knockdown with any dsRNA did not alter the number of periostial haemocytes, the number of non-periostial sessile haemocytes, melanin accumulation at the periostial regions or systemic infection intensity . However, knockdown of PGRP-LA-RC increased phagocytosis of GFP-E. coli in the periostial regions, which suggests that the RC splice form is involved in the phagocytosis response .PGRP-LC is the canonical activator of the IMD pathway , but PGRemocytes  and . Because of high sequence identity between JNK1 and JNK3, dsJNK1/3 simultaneously targeted both JNK genes. RNAi-based knockdown resulted in 33%, 44% and 31% reduction in mRNA abundance of JNK1, JNK3 and puc, respectively . Moreover, because the JNK pathway has also been implicated in the expression of TEP1 [TEP1, cec3 and def1 in JNK1/JNK3 and puc RNAi mosquitoes. Manipulating the JNK pathway did not have a clear impact on effector gene expression . The lack of clarity on how the JNK pathway controls the expression of TEP1, cec3 or def1 could be due to their co-regulation by other pathways, or a consequence of incomplete gene silencing of JNK1/3 and puc.We next tested whether the JNK pathway regulates periostial haemocyte aggregation. and JNK3 . Therefo of TEP1 , we measJNK1/3 and puc RNAi mosquitoes, relative to dsR)bla(Ap control mosquitoes (figures JNK1/3 and dsR)bla. Knockdown of puc, however, increased the number of periostial haemocytes 1.6-fold in uninfected mosquitoes, and 1.4- and 1.5-fold at 4 and 24 h following GFP-E. coli infection, respectively, relative to similarly treated dsR)bla. JNK1/3 RNAi mosquitoes showed a trend of fewer non-periostial sessile haemocytes than control mosquitoes regardless of infection status. Treatment with dspuc, however, increased the number of non-periostial sessile haemocytes for all treatments. This demonstrates that the JNK pathway positively regulates both periostial and non-periostial sessile haemocyte abundance.To assess whether the JNK pathway drives heart-associated immune responses, we compared the number and activity of periostial haemocytes in  figures  and 5. Reatments a,b. Knocsquitoes c. JNK1/3a; electronic supplementary material, figure S8). At 4 h following infection, the GFP-E. coli pixel area was similar regardless of dsRNA treatment. However, at 24 h following infection, periostial haemocytes in dspuc mosquitoes phagocytosed more GFP-E. coli than the dsR)bla. Melanin deposits were undetectable in uninfected mosquitoes and mosquitoes at 4 h post infection; however, at 24 h following GFP-E. coli infection, more melanin was present in dspuc mosquitoes than in dsR)bla enhanced phagocytosis by periostial haemocytes or (ii) higher bacterial proliferation in the haemocoel. To differentiate between these two scenarios, we quantified the systemic E. coli infection intensity and found that the bacterial intensity was similar regardless of dsRNA treatment (c). Therefore, this suggests that puc negatively regulates haemocyte adhesion and phagocytic activity, and demonstrates that the JNK pathway is a positive regulator of periostial immune responses.The difference in GFP-reatment c. Theref. 3Previous microarray and RNAseq analyses revealed genes and signalling pathways that are active in mosquito haemocytes –58, inclrel2, decreases infection-induced periostial haemocyte aggregation, whereas knockdown of the negative regulator of the IMD pathway, caspar, increases the number of periostial haemocytes. We hypothesize that the IMD pathway drives periostial haemocyte aggregation via two cascades that are also driving phagocytosis: the TEP1-TEP3-LRP1-CED6L and TEP4-BINT2-CED2L-CED5L pathways [TEP1 and TEP4 [rel2, bacteria proliferated uncontrollably during the later stages of infection. We hypothesize that knocking down rel2 initially suppresses periostial haemocyte aggregation, but as the infection progresses and the intensity increases, the necessity of periostial immune responses also increases, and this pressure recruits haemocytes to the heart and enhances their collective phagocytic activity. Overall, our data show that the IMD pathway drives periostial haemocyte aggregation during the early stages of infection and limits systemic infection intensity during the later stages of infection.The IMD pathway controls the production of antimicrobial peptides ,27, but pathways . The IMDand TEP4 ,31,68, aand TEP4 . Moreoveand TEP4 –71, and and TEP4 ,68,72–78puc, increases the number of periostial and adjacent sessile haemocytes. This suggests that the JNK pathway positively regulates haemocyte adhesion. Indeed, the Drosophila orthologue of mosquito JNK1 and JNK3, called basket, is involved in the formation of actin-rich and focal adhesion kinase-rich placodes in haemocytes [Drosophila, producing large lamellocytes that adhere and encapsulate parasitoid eggs [E. coli and melanin on the heart, suggesting that the JNK pathway positively regulates the phagocytosis and melanization responses. Indeed, JNK1 positively regulates melanization in mosquitoes that are refractory to malaria [JNK1 and JNK3 play in periostial haemocyte aggregation. However, simultaneous knockdown of both resulted in phenotypes that were opposite of puc knockdown, strongly suggesting that JNK1 or JNK3—or both—positively regulates periostial haemocyte aggregation.The JNK pathway modulates mosquito longevity, regulates oviposition, and limits infection with malaria parasites and viruses ,51,79–82emocytes . The JNKoid eggs . We foun malaria . We hypo malaria ,15. The  malaria ,80,86, a malaria ,87–89. ITEP1 [Drosophila, draper and the BINT2 orthologue (Integrin βν) function independently [Because both the IMD and JNK pathways induce the production of TEP1 ,33,68,80TEP1 ,35. Secoendently , and in endently ,67,70,86upd3a by periostial haemocytes, which is a ligand for the receptor of the JAK/STAT pathway [TEP1 [Bacteria, malaria parasites and fungal components all induce periostial haemocyte aggregation, and this process is structurally conserved across the entire insect lineage ,6,13. Th pathway . The Tol pathway ,93 and tay [TEP1 , which pay [TEP1 . Beyond ay [TEP1 . TherefoDrosophila, called Branchless, is expressed in the heart and pericardial cells, and regulates haemocyte differentiation in the lymph glands of the larval heart [Drosophila cardiac extracellular matrix (ECM) proteins, Pericardin and Laminin A, disrupts the formation of the cardiac ECM and lowers the number of haemocytes on the heart [To date, we have tested the involvement of several haemocyte-produced factors on periostial haemocyte aggregation. However, the heart itself may produce complementary factors that drive this immune response. For example, a fibroblast growth factor is highly regulated in the heart and periostial haemocytes. Its orthologue in al heart ,96. Morehe heart ,97. ThesDrosophila, transglutaminase negatively regulates the IMD pathway by (i) crosslinking the transcription factor, Relish, into a polymer and (ii) incorporating natural primary amines into the DNA binding site of Relish [A. gambiae, and found that TGase3—but not TGase1 or TGase2—negatively regulates periostial haemocyte aggregation during the early stages of infection and the sequestration of melanin by periostial haemocytes during the later stages of infection [TGase2 and TGase3 has infection-dependent effects on the heart rate [The mosquito genome encodes three transglutaminase genes, and our RNAseq experiment revealed that two of them are upregulated in the heart and periostial haemocytes following infection. In f Relish ,39. We rnfection . Moreoveart rate . CombineA. gambiae sheds light on how this medically important insect and other insects of societal importance fight the pathogens that invade them. Indeed, the TEP gene family, the Nimrod gene family  and the JNK pathway are conserved amongst insects [Heart-associated immune responses occur in insects from at least 16 different taxonomic orders, including insects that are of agricultural, urban and medical importance . Underst insects ,98–100,  insects –104. The. 4. 4.1Anopheles gambiae, Giles sensustricto , were maintained at 27°C, 75% relative humidity, and a 12 h : 12 h light : dark photoperiod [E. coli  and S. aureus (RN6390) were grown in Luria-Bertani (LB) and tryptic soy broth, respectively, at 37°C in a shaking incubator. Dilutions of the bacterial cultures were injected at the thoracic anepisternal cleft using a Nanoject III . The infection dose was determined by plating the cultures and counting the colony forming units (CFUs).toperiod . ExperimIn this study, a biological replicate is an independent experiment that uses mosquitoes from an independent egg batch. A technical replicate is an experimental resampling of a biological replicate. All data collected were included in the analysis; no data were excluded.. 4.2E. coli (approx. 75 779 CFUs) and (iv) infected by injecting 69 nl of S. aureus (approx. 39 451 CFUs). At 4 h after treatment, three tissues were isolated: (i) the heart with periostial haemocytes, (ii) haemolymph with circulating haemocytes and (iii) the entire abdomen containing all its internal organs and tissues . To isolate the heart, mosquitoes were bisected along the coronal plane in RNase-free PBS, and the heart with the periostial haemocytes was resected by severing the alary muscles and detaching it from the cuticle [Six-day-old mosquitoes were randomly divided into four groups: (i) naive (unmanipulated), (ii) injured by injecting 69 nl of sterile LB, (iii) infected by injecting 69 nl of GFP- cuticle . The abdTo isolate RNA from hearts or abdomens, samples were homogenized in TRIzol Reagent , extracted following the TRIzol protocol, and resuspended in Buffer RLT  with 2% 2-mercaptoethanol. The RNA was further purified using the RNeasy Micro Kit (Qiagen), DNase treated, and eluted in RNase-free water. To isolate RNA from circulating haemocytes, perfused haemolymph was collected in Buffer RLT and 2% 2-mercaptoethanol, and RNA was isolated using the RNeasy Micro Kit as above.a priori statistical method was used to pre-determine sample sizes. The number of RNAseq samples and biological replicates was determined prior to the initiation of the study, and was based on our determination that three biological replicates is a conservative approach to detect statistical differences (if any) in gene expression. Three biological replicates were conducted; each heart sample contained 72 hearts, each haemolymph sample contained perfusate from 108 mosquitoes, and each abdomen sample contained 36 abdomens.No The integrity and quantity of RNA was assessed on a 2100 Bioanalyzer  using an RNA 6000 Series II Nano kit for abdomen samples and a Pico kit for the heart and haemolymph samples. The library for sequencing was prepared using 1 µg of RNA and the NEBNext Ultra kit , according to the protocol for low-input samples. Library quality and concentration were assessed using a DNA 1000 Series II kit on the 2100 Bioanalyzer.. 4.3A. gambiae genome (AgamP4.7) by STAR [2 fold change ≥ 2 or log2 fold change ≤ –2 and the Benjamini–Hochberg adjusted p < 0.05. RNAseq results are presented in electronic supplementary material, dataset S1, which includes read counts and log2 fold changes. Using fourfold expression difference as the criterion, together with experimental replication and statistical validation, is a conservative approach that is proven to yield accurate and reliable results [https://www.ncbi.nlm.nih.gov) as SRA data PRJNA730047.All 36 samples—four treatments, three tissue types and three biological replicates—were sequenced across three lanes on an Illumina HiSeq 3000  at Vanderbilt University's Vantage facility. Reads were mapped to the  by STAR ,108. The by STAR . Genes w results . RNAseq . 4.4rel2, caspar, PGRP-LA, JNK1 and JNK3, and puc. Anopheles gambiae cDNA was amplified by PCR using gene-specific primers with T7 promoter tags . dsRNA was synthesis using the MEGAscript T7 Kit (Applied Biosystems) as described [R)bla containing the pET-46 plasmid as template  [Double-stranded RNA was synthesized for escribed ,15. As aown, NJ) ,15.E. coli (approx. 16 528 CFUs). Injured mosquitoes were not included in the experiments because injury does not induce periostial haemocyte aggregation [TEP1, cec3 and def1 was determined by qPCR [CT−ΔΔ method, with RpS7 as the reference and RpS17 as a control [Two- or three-day-old mosquitoes were intrathoracically injected 300 ng of dsRNA to initiate systemic gene silencing. Four days later, mosquitoes were divided into two groups for phenotypic analyses: (i) uninfected and (ii) infected with GFP-regation –6. RNAi  by qPCR . Briefly by qPCR ,15. qPCR control . Two to . 4.5Haemocytes were labelled with the Vybrant CM-DiI Cell-Labeling Solution (Invitrogen) as we described . Briefly. 4.6Z-stacks for bright field, red fluorescence (haemocytes), green fluorescence (GFP-E. coli) and blue fluorescence (nuclei) were acquired using a linear encoded Z-motor. Specific channels were selected and all images within a stack were combined into a two-dimensional image using the extended depth of focus (EDF) function.Specimens were imaged on a Nikon Eclipse Ni-E compound microscope connected to a Nikon Digital Sight DS-Qi1 camera and Advanced Research NIS Elements software . . 4.7Z-stack [R)bla. Data for RNAi phenotypic experiments are from individual mosquitoes, which were sampled across three to four biological replicates. The exception is experiments for PGRP-LA, where mosquitoes were sampled across two biological replicates.Haemocytes, labelled with both CM-DiI and Hoechst 33342, were counted manually by examining all images within a Z-stack . A cell Z-stack . PeriostZ-stack ,8. Data . 4.8E. coli at the periostial regions was calculated by measuring the area of pixels with intensities above a threshold that distinguished GFP-E. coli from background fluorescence [R)blabla(Ap mosquitoes as the reference. For rel2 and caspar RNAi mosquitoes, data were first log2 transformed to achieve normality, and then analysed by two-way ANOVA.Mosquitoes that were infected with tetracycline resistant GFP-


The auditory pathway is the main target for high levels of blood sugar. Increased glucose in diabetic patients can disrupt the auditory system physiologically and anatomically. The present study aimed to examine the prevalence of hearing loss in patients with type 2 diabetes. A total of 94 patients with type 2 diabetes, aged from 40 to 80 years, were selected randomly in the present descriptive cross-sectional study for pure tone audiometry (PTA), speech discrimination score (SDS), and speech reception threshold (SRT) tests. Accordingly, patients with a hearing threshold larger than or equal to 25 dB were considered hearing-impaired according to the PTA test. In addition, the patients' speech discrimination score was measured using a list of monosyllabic words with an intensity of 40 dB or more than the SDS test. However, in the SRT test, the patients' superficial speech comprehension threshold was measured using a list of two-syllable words. Most diabetic patients had hearing loss in both right and left ears based on the PTA and SRT tests. However, they did not have hearing loss in both ears according to the SDS test. There was no correlation between the PTA, SRT, and SDS tests and blood creatinine levels in both ears (p>0.05). Nevertheless, the right ear had a positive correlation with systolic blood pressure only in the PTA test (p<0.05). The difference between the two groups of men and women with type 2 diabetes in the hearing level in the right and left ears was not statistically significant. Hearing loss is a common deficiency in diabetic patients. In addition, it seems that diabetes is an independent risk factor for the hearing loss level. Hearing loss is one of the most common disabilities in the United States. The prevalence of this defect is more than 16% in the adult population and two-thirds of the population over 70 . The rolThe auditory pathway has high metabolic activity, so it is the target organ for the pathogenesis of hyperglycemia and diabetes . High blSeveral mechanisms have been suggested for the development of hearing loss in diabetic patients, including microangiopathy, neuropathy, and oxidative stress damage , 11. DiaBainbridge et al. (2016) examined risk factors for hearing loss in diabetic patients. Their results showed that alcohol consumption, smoking, chronic renal failure, and high triglycerides were significantly associated with hearing loss . Nwosu eThe auditory pathway is susceptible to metabolic changes due to hyperglycemia, so diagnosing hearing impairment in diabetic patients is important. Thus, evaluating the prevalence of hearing loss in diabetic patients is effective in finding a way to decrease hearing loss among them. Therefore, this study aims to assess the prevalence of hearing loss in diabetic patients.The target population in the present descriptive cross-sectional study was patients with type 2 diabetes referred to the endocrinology department and diabetes clinic of Ali Ibn Abi Taleb Hospital, Rafsanjan, in 2018. Accordingly, 9,206 patients entered this study. In the end, 94 patients with type 2 diabetes , aged frThe criteria for diagnosing diabetes in people were having a fasting blood sugar of more than 125 mg/dL and the glucose tolerance test (GTT) ≥200 or 6.5 ≤HbA1c . The patPatients underwent audiometric testing using the PTA, SDS, and SRT tests. In the PTA test, patients were subjected to sounds at different frequencies, and their hearing thresholds of air transmission and bone transfer were measured separately using an audiometer. Accordingly, patients with a hearing threshold higher than or equal to 25 dB were considered to have hearing impairment in the PTA test. In addition, the patients' speech discrimination score was measured using a list of monosyllabic words with an intensity of 40 dB or higher in the SDS test. Finally, in the SRT test, the patients' superficial speech comprehension threshold was measured using a list of two-syllable words .The data were analyzed using SPSS V21.0. The groups were compared using parametric and non-parametric statistical methods. Student's t-test, chi-square test, one-way ANOVA, and Tukey's post hoc test were used to compare the data between different groups. The data were presented as mean (SD). In addition, differences were considered statistically significant at P<0.05 and P<0.01.The patients' mean age was 61.54±10.16. The data showed that most patients (63 patients) were female and housewives; 25 were employees, 5 were workers, 14 were farmers, and 50 were housewives . The creHowever, the results showed a significant difference in terms of the age of all groups and different hearing loss levels (p<0.01). Also, there was no significant difference between the two genders in terms of the prevalence of hearing loss (p>0.05) . Creatin2=1.40) , with high diastolic blood pressure (p>0.05). Besides, there was no significant difference between different types of medicines in terms of the prevalence of hearing loss (p>0.05) . Most of2=1.40) .According to the PTA test, most patients had hearing loss in both right and left ears . Similarly, most of the patients had hearing loss in both right and left ears , according to the SRT test. Nevertheless, the SDS test showed that most patients had no hearing loss in the right and left ears  . The rigYounger patients aged less than 65 had significantly higher hearing levels in both right and left ears than patients older than 65 years . Female The results of the present study showed that most of the patients with type 2 diabetes (35.1%) had mild hearing loss. Besides, our findings showed that most patients with hearing loss were older than 65 years. In addition, patients under 65 years old had better hearing in both left and right ears. Furthermore, most of the patients with hearing loss were male. However, there was no significant difference between the two genders in the prevalence of hearing loss. Moreover, there was no significant difference between diabetic men and women in hearing loss in the right and left ears. Similarly, Ghanaati et al. reported no significant difference between males and females , with thThere was no significant difference in the prevalence of hearing loss between the two groups in the present study in terms of creatinine levels less than 1.2 and more than 1.2. Furthermore, the patients' scores in the PTA, SRT, and SDS tests in both right and left ears were not related to blood creatinine levels. Samelli et al. (2017) evaluated patients' scores in PTA, SDS, and SRT tests in the right and left ears and compared them with the mean of patients' blood creatinine levels. Their results showed no significant relationship between creatinine levels and hearing loss test scores in both ears, which is consistent with our study .Bainbridge et al. (2016) evaluated risk factors in diabetic patients. Their findings showed that alcohol consumption, smoking, chronic renal failure, and high triglyceride levels were significantly associated with hearing loss in diabetic patients. In addition, low income was reported as a possible causative factor for hearing impairment . InconsiThe findings of the present study showed that systolic blood pressure was high in diabetic patients with mild hearing loss. Besides, diastolic blood pressure was high in diabetic patients with mild, moderate, and severe hearing loss as well as in those with no hearing loss. However, this difference was not statistically significant. Samelli et al. reported no association between the hearing threshold with age, gender, and hypertension. They concluded that the mean SRT and SDS in diabetic patients were 16.2±9.1% and 94.9±6.3%, respectively .Mashhadi et al. (2015) examined the relationship between diabetes and hearing loss. Audiometric findings at different frequencies in diabetic and control groups showed no significant relationship between diabetes and hearing loss . HoweverConsistent with our findings, Zivkovic-Marinkov et al. 2016) reported that the hearing threshold was significantly higher in diabetic patients than in healthy cases. Moreover, there was no difference among the patients in hearing loss in terms of different types of medications 16 report. The typOur study showed that mild to moderate hearing loss in diabetic patients was bilateral, with patients with a severe hearing loss having unilateral defects. Moreover, there was a significant difference between hearing loss types in diabetic patients. Accordingly, employed patients had better hearing in both right and left ears. In addition, housewives and employed patients had higher hearing loss in the right and left ear, respectively, than other patients, with no statistically significant difference. Hearing loss increased in farming patients, which could be due to their higher exposure to agricultural machines causing strong noise pollution.Our findings showed that 36.2% of diabetic patients suffered moderate to severe hearing loss. Also, there was a positive relationship between aging and hearing loss in diabetic patients. Accordingly, it seems that diabetes is a direct risk factor for hearing loss.


There was a significant difference between the change of BC over the years and the HISQUI, as well as the number of years after implantation and the HISQUI. On average, per dB decrease in BC, the HISQUI decreases by 1.4 points, and every year after implantation the HISQUI decreases by 2.7 points. Conclusions: The aided threshold in free field and speech understanding in noise improved significantly with VSB. An increase over time of BC thresholds was observed as well as a decrease in HISQUI score. This decrease in BC thresholds over time may be due to presbycusis. Therefore, monitoring of these patients over time should be considered to discuss alternative hearing rehabilitation measures in a timely manner.Background: To evaluate the long-term audiological outcomes combined with the Hearing Implant Sound Quality Index (HISQUI) after Vibrant Soundbridge (VSB) implantation. Methods: Prospective recall cohort study of patients who received a VSB in a tertiary academic medical center between 1996 and 2017. Air conduction (AC) and bone conduction (BC), sound field thresholds in aided and unaided conditions, and speech discrimination in noise (Oldenburger sentence test) were measured. Postoperative results were compared with preoperative audiograms. Furthermore, the HISQUI was evaluated. Results: Ten patients (eleven implants) were included, the mean follow up period was nine years. The mean AC threshold preoperatively was between 63 and 70 dB, and the BC was between 38 and 49 dB from 500 to 4000 Hz. In the free-field audiogram, the mean threshold was between 61 and 77 dB unaided vs. between 28 and 52 dB in the aided condition. The average signal to noise ratio (SNR) in the Oldenburger sentence test in the unaided condition was 10 dB ± 6.7 dB vs. 2 dB ± 5.4 dB in the aided condition. Three patients reported a good to very good hearing result, four patients a moderate, and three patients a poor hearing result. There was a significant association between the years of implantation and the HISQUI ( It has been shown that an active middle ear implant is an alternative for patients with conductive or mixed hearing loss, who do not benefit from conventional hearing aids or cannot wear a conventional hearing aid because of aural atresia, chronic inflammations of the outer ear canal, severe allergies, or inflammations arising from wearing hearing aids. One commercially available active middle ear implant is the Vibrant Soundbridge (VSB) . The VSB consists of two primary components: the audio processor (AP) as an external unit, and the surgically implanted vibrating ossicular prosthesis (VORP). The AP consists of a microphone, signal processing electronics, a telemetry coil, and a battery to receive and process incoming acoustical signals transdermal to the implanted VORP. The VORP consists of three further components: the implanted receiver unit, the conductor link, and the Floating Mass Transducer (FMT) ,2. The first clinical trials to investigate results after VSB implantation in Europe and the United States started in 1996 . Since tThere is no standardized questionnaire to evaluate patient satisfaction for active middle ear implants. For cochlear implant users, the Hearing Implant Sound Quality Index (HISQUI19) was developed and validated to evaluate the self-perceived level of auditory benefit in cochlear implant patients. The questionnaire consists of 19 questions describing every day listening situations . As a usThere are relatively few studies reporting on the audiological outcomes together with subjective benefits for VSB . TherefoThe study protocol was approved by the local Ethical Committee (KEK-ZH No. 2016-01593). All patients received an implantation of an active middle ear implant (Soundbridge) between 1996 and 2017 at the ENT Department, University Hospital of Zurich, Switzerland. All study participants were ≥18 years old and provided written informed consent prior to the examinations. All audiometric tests were performed following standard procedures in accordance with ISO 8253-1. An AC pure-tone audiogram for each side with headphones was conducted for 500, 1000, 2000, 3000, and 4000 Hz at two time points: (1) before surgery, (2) at the time of follow up with the middle ear implant device turned off. The results were compared to detect changes in the hearing threshold. At the time of follow-up, a free field audiometry test was performed with the same frequencies with the stimulus coming from a loudspeaker 1 m in the front of the participants. First, the active middle ear implant was turned off (unaided), then we performed the test with the device turned on (aided). To rate the benefit of the middle ear implant, the two tests were compared. The hearing tests were measured with appropriate contralateral masking through calibrated headphones.Speech discrimination was evaluated using the Oldenburger sentence test in noise (OLSA) . The OLSTo evaluate the subjective hearing benefit of the implant, the HISQUI19 questionnaire was used . This inp < 0.05. Statistical analysis was performed with R version 3.2.0 . Graphs and figures were constructed with R and GraphPad Prism (8.3.1). Data of pure tone audiograms, as well as free-field audiogram measurements and signal-to-noise ratios, were reported as mean ± standard deviation (SD). Linear regression was used to test the associations between the subjective hearing benefit and the audiometric parameters of inner ear function. The level of statistical significance was set at Thirty-seven patients have been implanted with VSB in our tertiary academic hospital center between 1996 and 2017. Ten patients could not be contacted because they moved away without leaving their contact data, nine refused further tests, five were under 18 years old or not able to give informed consent due to their mental state, two did not use the implant anymore, and one patient had passed away (dropout rate of 73%). Ten implanted patients  were included in this study. The demographics of included patients are shown in The average AC threshold preoperatively was 63 dB ± 20 dB SPL at 500 Hz, 65 dB ± 19 dB SPL at 1000 Hz, 64 dB ± 17 dB SPL at 2000 Hz, and 70 dB ± 19 dB SPL at 4000 Hz. The average BC threshold preoperatively was 39 dB ± 15 dB SPL at 500 Hz, 38 dB ± 19 dB SPL at 1000 Hz, 49 dB ± 14 dB at 2000 Hz, and 42 dB ± 16 dB at 4000 Hz . The average unaided AC threshold postoperatively was 66 dB ± 22 dB SPL at 500 Hz, 70 dB ± 24 dB SPL at 1000 Hz, 72 dB ± 22 dB SPL at 2000 Hz, and 77 dB ± 22 dB SPL at 4000 Hz. The average BC threshold postoperatively was 35 dB ± 18 dB SPL at 500 Hz, 41 dB ± 19 dB SPL at 1000 Hz, 57 dB ± 19 dB at 2000 Hz, and 50 dB ± 21 dB at 4000 Hz. Pre- and postoperatively, hearing thresholds did not change significantly. For AC, they were 3 dB for 500 Hz, 5 dB for 1000 Hz, 8 dB for 2000 Hz, and 7 dB for 4000 Hz. For BC, the difference was −dB for 500 Hz, 3 dB for 1000 Hz, 8 dB for 2000 Hz, and 8 dB for 4000 Hz .p = 0.008), 28 dB ± 6 dB at 1000 Hz (p = 0.002), 29 dB ± 11 dB at 2000 Hz (p = 0.003), and 52 ± 15 dB at 4000 Hz (p = 0.034). The average gain in dB SPL with the device active (aided) in the free-field audiometry was 23 dB ± 17 dB SPL at 500 Hz, 35 dB ± 17 dB at 1000 Hz, 36 dB ± 24 dB at 2000 Hz, and 25 dB ± 26 dB at 4000 Hz (The average threshold with the device turned off (unaided) in the free-field audiogram was 61 dB ± 17 dB SPL at 500 Hz, 63 dB ± 19.5 dB at 1000 Hz, 65 dB ± 20 dB at 2000 Hz, and 77 dB ± 23 dB at 4000 Hz. The average threshold with the device turned on (aided) was significantly lower: 39 dB ± 8 dB at 500 Hz ( 4000 Hz . p < 0.01) and showed no difference in 500 Hz, 1000 Hz, and 4000 Hz (The difference between BC postoperatively and the free-field audiogram with the device turned on (aided) was 28 dB in 2000 Hz   in the Oldenburger sentence test in the unaided condition (where patients did not wear the processor) was 10 dB ± 6.7 dB. Aided, the SNR was 2 dB ± 5.4 dB. There was a significant difference of −8 dB SNR with the device (< 0.001) . One patTwo patients reported >111 points (very good hearing result), one patient had 103 points (good), four patients were between 69 and 88 points, which corresponds to a moderate result, and three patients reported to scored less than 59 points, which corresponds to a poor result. The mean HISQUI was 83 points (moderate).p = 0.023, adjusted R2 = 0.39)  . p = 0.013) (see p = 0.519), nor in the unaided (p = 0.459) condition was associated with the HISQUI. Additionally, there was a further significant association between the number of years of implantation and the HISQUI. On average, every year after implantation, the HISQUI decreases by 2.7 points  see . NeitherThis study evaluated the long term outcome after VSB implantation. A review of the literature indicates that hearing threshold is not affected in the middle- to long-term follow up after VSB implantation compared to the opposite non-operated ear ,11. VariA very important additional factor in determining the outcome of an intervention is patient satisfaction. Since no specific questionnaires for active middle ear implants exist, the HISQUI questionnaire was used because it has proven to be user-friendly and has been used to determine subjective auditory benefit in other implants, such as the Bonebridge hearing implant . DespiteA direct comparison to other studies is difficult, because various questionnaires are used by different authors. Ihrler et al. used the Glasgow Benefit Inventory (GBI) and compared patients with an active middle ear implant to a matched population with conventional hearing aids . ZwartenThe age of the patient seems to affect speech recognition. Von Gablenz and Holube  investigThere was a correlation between the average change of BC threshold  and the HISQUI. The higher the deterioration, the less the subjective hearing implant sound quality. The influence of higher complex central processing that changes with age may be the reason for these findings . There are limitations of our study. For a variety of different reasons, we were only able to include 10 out of 40 patients. Therefore, we did not differentiate the subjects into further subgroups such as sensorineural hearing loss vs. mixed hearing loss, and did not differentiate different coupler systems. We did not evaluate individual reasons for study participation, or soft factors such as how many times the patients visited their hearing professionals to adjust the hearing aids. Due to the limited number of included patients, the statistical analysis and interpretation of data need to be interpreted with care. Furthermore, the time of follow-up was variable and ranges from several months to several years. Finally, the HISQUI was only documented once during the follow-up at individual time points.Aided thresholds with VSB in free field improved significantly for a follow-up of up to 20 years. Additionally, speech understanding in noise improved significantly. However there was a decay of the subjective sound quality detection (HISQUI) over time, and an increase in BC thresholds. Most likely, the increased BC threshold is due to presbycusis, causing patients to have issues with audibility and therefore decreasing the benefit of VSB. Patients with VSB should be counselled that they may fall out of the indication range with time. Therefore, monitoring of these patients over time should be considered to discuss alternative hearing rehabilitation measures such as cochlear implants if required in good time. However, due to the low number of included patients, investigations on larger patient groups are required to confirm this assumption.


Enhancing the luminescence property without sacrificing the charge collection is one key to high-performance organic solar cells (OSCs), while limited by the severe non-radiative charge recombination. Here, we demonstrate efficient OSCs with high luminescence via the design and synthesis of an asymmetric non-fullerene acceptor, BO-5Cl. Blending BO-5Cl with the PM6 donor leads to a record-high electroluminescence external quantum efficiency of 0.1%, which results in a low non-radiative voltage loss of 0.178 eV and a power conversion efficiency (PCE) over 15%. Importantly, incorporating BO-5Cl as the third component into a widely-studied donor:acceptor (D:A) blend, PM6:BO-4Cl, allows device displaying a high certified PCE of 18.2%. Our joint experimental and theoretical studies unveil that more diverse D:A interfacial conformations formed by asymmetric acceptor induce optimized blend interfacial energetics, which contributes to the improved device performance via balancing charge generation and recombination. High-performance organic solar cells call for novel designs of acceptor molecules. Here, He et al. design and synthesize a non-fullerene acceptor with an asymmetric structure for diverse donor:acceptor interfacial conformations and report a certificated power conversion efficiency of 18.2%. However, the PCEs remain lower than those of inorganic materials, e.g. silicon (PCE up to 26.7%) or GaAs (29.1%)5, as a result mainly of the larger open-circuit voltage (Voc) loss that penalizes organic solar cells (OSCs) (Voc loss ~0.55 eV for the state-of-the-art PM6:Y6 systems)11.Organic photovoltaics (OPV) is an emerging technology that presents great potential in terms of renewable energy source, due to specific advantages of organic materials, such as flexibility, low cost, selective absorption for visible transparency, vivid colors, etc. With comprehensive efforts in molecular design, morphology control, interface engineering, and device architecture, the current record power conversion efficiency (PCE) has reached 19%, which pushes OPV to the edge of commercializationVoc loss12. This is a major advance compared to fullerene-based OSCs, whose large Voc loss is typically related to a much lower CT-state energy compared to the LE-state energy15. In addition, non-radiative decay can be significantly reduced in NFA-based devices17. Overall, the best performance from first-generation NFAs, e.g., IT-4F or its derivatives, reaches over 15% PCE18, while the emergence of Y6 and its derivatives has brought PCEs over 18%. Y6 has a banana shape and a rather porous three-dimensional (3D) packing pattern, which results in remarkable optoelectronic properties, including a low exciton-binding energy and ambipolar charge transport ability with high carrier mobilities20.The recent development of non-fullerene acceptors (NFAs) has produced charge-transfer (CT) state energies close to that of the strongly absorbing (and emitting) local-exciton (LE) state on the donor and/or acceptor, which has contributed to significantly reduced 21.When considering the evolution of the acceptor molecular symmetries (from the fullerene family to the ITIC family and the Y6 family), it is most interesting to realize that the degree of molecular symmetry gradually reduces, from the ball-shaped  fullerenes, to ITIC molecules with a largely coplanar core and inversion symmetry, to Y6 molecules with a banana-shape and a helical chiral structure. In this context, the logical next step is the development of asymmetric acceptors, a strategy that is getting increasing attention, as reported by Li et al. 26. We summarize the recently reported Y-series asymmetric NFAs in three categories: (i) Y-series NFAs with asymmetric backbones24; (ii) Y-series NFAs with asymmetric side chains23; (iii) Y-series NFAs with asymmetric terminals28  of 10−3 and EQEPV approaching 80%. Further, adding an asymmetric molecule as the third component will induce similar effect as that in binary blend, i.e. the more diverse D:A interfacial conformations over the symmetric ones. As a result, the impact of exploiting the asymmetric BO-5Cl molecule as third component also manifests in ternary blends with PM6 and BO-4Cl, as it leads to high-performance devices with a PCE of 18.56% (certified as 18.2%), which is among the highest certified PCE values.Here, based on the design of the BO-5Cl molecule see Fig.  that dis27. These molecules represent the current state-of-the-art in terms of performance of Y-series NFAs30. The synthetic routes of the four acceptors are illustrated in Supplementary Fig. 27. By adopting 2- malononitrile (T-Cl) on one side while maintaining IO-4Cl on the other side, we obtain the BO-5Cl molecule that has a lower ionization potential  level). The detailed synthesis protocols and structure characterizations can be found in Supplementary Figs. The asymmetric molecular structures of BO-5Cl and BO-6Cl are shown in Fig. 1) has a significant intra-molecular CT character, with the hole localized on the BTP core and the electron delocalized on both end groups. A similar intra-molecular CT description is obtained for BO-5Cl, however, with the major difference that only the T-Cl end group is involved in the excitation; the S1 state of BO-5Cl is characterized by a very large state dipole moment of about 15 D, to be compared to 4 D calculated for S1 in BO-4Cl  spectra when casting these NFAs into thin films Fig. . For insCyclic voltammetry (CV) was carried out to measure the state energies; the measured ionization potentials and electron affinities are displayed in Fig. 31. We first analyzed the molecular packing characteristics in single crystals of BO-4F, BO-4Cl, and BO-5Cl. Details regarding single-crystal growth can be found in the Supplementary Fig. 31) but also between overlapping cores (as found in Y6)32. The numerical values highlighted in Supplementary Fig. In addition to their molecular structures, the packing behavior of the NFAs has also been demonstrated to play a key role in determining their optoelectronic properties and OSC performance33. A similar picture is obtained for BO-4Cl  related to electron transport are significant (larger than 50 meV) in both BO-4Cl and BO-5Cl crystals  in the out-of-plane direction and at qxy = 0.390 Å−1 (d = 16.0 Å) in the in-plane direction are observed, which indicates a dominant face-on orientation of the molecular stacking (detailed packing parameters are collected in Supplementary Tables qz = 1.78 Å−1 (d = 3.53 Å) and qr = 0.389 Å−1 (d = 16.2 Å) in the single crystal are preserved in the PM6:BO-4Cl blend film. It can be anticipated that the molecular packing in the pure acceptor domains of the blend film is similar to the single-crystal stacking pattern9. This means that the transport networks discussed above for the acceptor films are also expected to be inherited into the D:A blends9.Grazing-incidence wide-angle and small-angle X-ray scattering (GIWAXS/GISAXS) characterizations were performed to investigate the morphology of the pristine acceptor films Fig.  and 13 a35. The acceptor domain sizes (2Rg) of the five blends are similar (9–12 nm), all within the appropriate range for efficient exciton dissociation; the intermixing domain size (ξ) of PM6:BO-5Cl is the smallest, which will facilitate the charge collection from this region  between those of the parent binary blends , illustrating that the ternary system combines the fast exciton dissociation of PM6:BO-4Cl with the long diffusion process of PM6:BO-5Cl  spectroscopy measurements were performed to investigate the interfacial exciton dissociation, which is one of the critical processes in OSCs  measured by the hole transfer process matches well the PL lifetimes of the acceptors, which suggests that longer exciton lifetimes can extend the exciton diffusion lengths and thus the hole transfer times.To gain a deeper understanding of the hole transfer dynamics from the acceptor excited state to the donor in different systems, we also measured the TA spectra and extracted the kinetics for PM6 blended with BO-4F and BO-6Cl  as a function of the energetic difference of the D and A ionization potentials ΔIPD–A for all blends shown in Fig. τ1 represents the hole transfer rate; kB, the Bolzmann constant; T, the temperature; V, the electronic coupling between initial and final states; λ, the reorganization energy obtained by means of DFT calculations  or by ΔIPD–A as a simple approximation39. Importantly, since the reorganization energies in these blends are calculated to be very small (λ ~ 0.09–0.10 eV), the hole transfer rates can remain fast for a ΔIP value as small as, or slightly higher than 0.02 eV; this is a much lower value than the value of ~0.3 eV often quoted as required for the energy offset to drive efficient exciton dissociation17. Overall, the Marcus theory model describes well our TA kinetics results.Interestingly, the exciton dissociation rate constants . However, due to the contribution coming from exciton diffusion, it takes more than 100 ps to complete the exciton dissociation process44. We note that, in addition to exciton dissociation and exciton diffusion, the TA data might also be affected by other processes such as back electron/hole transfer from CT to LE state13, formation of excimers, and decay of CT states to local triplet states45. Therefore, in order to shed more light on exciton kinetics and examine the effect of different D:A packing configurations  is about 0.2 in both acceptors. In PM6:BO-4Cl and PM6:BO-5Cl, the I2/I1 ratios significantly increase. The energy difference between the first and second peaks in PM6:BO-5Cl resembles that of the acceptor, while in PM6:BO-4Cl it increases to 170 meV. These results strongly suggest that the second EL peak in both blends contains a significant contribution from the CT states. The EL results show that in both blends the I2/I1 values decrease with a temperature increase, which confirms the major contribution of the CT states to the second EL peak in the blends. Overall, the low-temperature EL results are consistent with an LE-CT energy difference that does not exceed 160 meV in PM6:BO-5Cl, while it is somewhat larger than 170 meV in PM6:BO-4Cl.In order to get a better assessment of the ΔPM6 Fig. . The EL J–V) curves, with the corresponding photovoltaic parameters summarized in Fig. Voc (0.833 V) and Jsc (26.04 mA cm−2) values of the PM6:BO-4F-based devices are almost unchanged, except that the FF increases from 0.740 to 0.772 due to the improved crystallinity29; accordingly, the PCE improves from 16.07% to 16.73%. For the PM6:BO-4Cl based devices, the Voc and FF values slightly increase to 0.841 V and 0.794, respectively, yielding a best PCE of 17.43%. For the asymmetric BO-5Cl and BO-6Cl based OSCs, their band gaps are enlarged  with solar spectra or the poor Jsc.The performance of the OSC devices based on PM6:NFA blends was examined by using a conventional device architecture consisting of ITO/PEDOT:PSS/active layer/PFN-Br/Ag. Details of device fabrication and characterizations are provided in the “Methods” section and Supplementary Tables Jsc in the former devices. The photovoltaic bandgaps (EgPV) are estimated to be 1.46, 1.47, 1.45, 1.56, 1.55, and 1.44 eV for PM6:Y6, PM6:BO-4F, PM6:BO-4Cl, PM6:BO-5Cl, PM6:BO-6Cl, and PM6:BO-4Cl:BO-5Cl, respectively, based on the method developed by Rau et al. , we find good correspondence between the integrated Jcal values and the Jsc from the J–V curves  coming from (i) charge recombination from the unavoidable black body radiation , (ii) non-ideal radiative decay , and (iii) non-radiative decay (∆E3)50. A detailed Eloss analysis was carried out as shown in Fig. Eloss analysis can be found in the Supplementary Fig. E1 due to the similar energy gaps of the five NFA-based devices. For the non-ideal radiative loss, PM6:Y6, PM6:BO-4F, PM6:BO-4Cl, PM6:BO-5Cl, and PM6:BO-6Cl exhibit different values of 0.042, 0.033, 0.042, 0.052, and 0.057 eV, respectively. The somewhat larger non-ideal radiative loss of the asymmetric acceptors can be attributed to the greater variation in molecular packing configurations between the asymmetric molecules in the amorphous state and/or between the donors and the acceptors, as shown by the DFT calculations.The major sources of EL results , China. This result is among the very highest reported certified PCEs to date. The introduction of asymmetric BO-5Cl results in more diverse D:A intermolecular conformations, which will balance the charge generation and charge recombination. Besides of the dilution effect6, the ternary blends (polymer donor with two different acceptors) could also construct diverse molecular conformations when an asymmetric molecule is added as the third component, which might show the similar effect as the asymmetric molecule appearing in the binary blend. Our findings validate the rationale of using asymmetric acceptors to achieve high luminescence and faster charge transfer for high-performance ternary-blend organic photovoltaic devices.Remarkably, the highly luminescent behavior of BO-5Cl can be exploited in other systems, specifically by making BO-5Cl a component in a ternary blend. For example, when blending 20% wt BO-5Cl into PM6:BO-4Cl, the charge recombination centers remain at the PM6:BO-4Cl interface, since the EL spectra of the ternary device keep a shape similar to that in PM6:BO-4Cl  and charge recombination (Voc or Voc loss) formed by more diverse D:A intermolecular conformations. Also, we discovered that the high-luminescence behavior can be transferred into other systems by developing ternary blends. In this instance, a high PCE of 18.56% is obtained (certified as 18.2%), which is due to a major reduction in Voc loss. These devices are among those with the very highest reported certified PCE values. Our joint experimental and theoretical efforts underline that the origin of the efficiency of organic solar cells based on asymmetric acceptors lies in the duality of the specific packing configurations and electronic characteristics at the D:A interfaces, which contribute to a better balance between charge generation and recombination in both binary and ternary active layers.We have demonstrated that the use of asymmetric acceptors offers a remarkable balance between charge transfer and charge recombination. We fabricated highly luminescent, efficient organic photovoltaic devices based on the PM6 donor and the BO-5Cl asymmetric acceptor, which is due to a better balance between the charge generation (EQE and −1 (PM6:acceptor = 1:1.2) chloroform solution at 3000 rpm for 30 s with different additives. Detailed device fabrication conditions are summarized in Supplementary Tables −1 PFN-Br in methanol. Finally, the Ag (100 nm) electrode was deposited by thermal evaporation. The device was completed with an active area of 0.0925 cm2, as defined by the overlapping area of ITO and Ag.Organic solar cells were fabricated on glass substrates commercially pre-coated with a layer of indium tin oxide (ITO), based on the inverted structure: ITO/PEDOT:PSS/active layer/PFN-Br/Ag. Before fabrication, the substrates were consecutively pre-cleaned by an ultrasonic bath of detergent, deionized water, acetone, and isopropanol, and then treated in an ultraviolet ozone generator for 15 min. Then, a thin PEDOT:PSS (Baytron P AI4083) layer was spin coated onto the substrates at 4500 rpm (~20 nm thick) for 30 s and annealed at 170 °C for 20 min. The substrates were placed into a glovebox and all active layers were spin coated from 17.6 mg mLJ–V) curves of OSCs were measured on an Enlitech SS-F5-3A solar simulator under AM 1.5 G illumination, whose light intensity was calibrated by a standard Si solar cell at 100 mV cm−2. Devices were tested in N2-filled glove box. The scan direction is −0.2 to 1.2 V, with a scan step of 0.01 V and dwell time is 1 ms. The EQE data were measured by a Solar Cell Spectral Response Measurement System . All of the devices mentioned were tested through a shadow mask with an area of 0.05979 cm2 .The current density–voltage (Further information on research design is available in the Supplementary InformationSolar Cells Reporting Summary


A new era has arrived in auditory rehabilitation with the introduction of new technologies such as electroacoustic stimulation (EAS). EAS is indicated for patients with residual hearing at low frequencies and severe or profound hearing loss at high frequencies. These patients have no indication for conventional cochlear implant and have difficulties in adapting to individual sound amplification devices. Preservation of hearing is vital in this process; the surgical technique must be based on this concept.To present the cochlear implant surgical technique with MED-EL Mand FlexEAS to preserve hearing in patients with hearing loss at high frequencies and to maintain low frequency hearing. We are the first institution to carry out this treatment in Brazil.A case report of the surgical technique carried out in four patients; the procedure was carried out by the cochlear implant group of a specialized clinical hospital.The procedures were successful and uneventful.We described the technique used at our institution for implants using EAS; the surgical technique is complex and includes steps for preservation of hearing. Just over a decade ago patients with sensorineural hearing loss had few options for auditory rehabilitation; there were hearing aids or sound amplification devices, electrical stimulation of the cochlear nerve (cochlear implants)These patients often present sloping hearing loss where low frequency thresholds  are preserved and higher frequency thresholds are lower, with severe or profound hearing loss at middle and high frequencies. A new form of therapy arose as the concept of auditory preservation developed and technological advances were made in cochlear implants, namely electric acoustic or hybrid stimulationKnowing about the diseases that cause hearing loss is fundamental to help patients by facilitating and speeding auditory rehabilitation processesChristian Von Ilberg developed the concept of electric acoustic stimulation (EAS) in 1999 The possibility of preserving residual hearing following cochlear implant surgery arose with the work of Lenarz; these patients respond better to hybrid stimulation of hearing – electric (cochlear implant) and acoustic (hearing aid)Several surgical techniques for preserving hearing have been describedThe intrinsic features of electrodes are important for reducing intracochlear trauma – the shape, length, and flexibility of the electrode arrays also help attain better resultsEAS electrode. We describe the surgical technique for EAS with the MED-EL Mand FlexEAS model, carried out by the Implantable Prostheses and Cochlear Implant Group, Otology Unit, of a tertiary care hospital, from March 2010 to May 2011.The purpose of this study was to present our experience of four patients that underwent cochlear implant surgery with the MED-EL Flex1Serial audiometry;2Free field audiometry;3Otoacoustic emissions (OAE) testing;4Speech perception test;5Brainstem auditory evoked potential (BAEP);6Computed tomography of the temporal bones;7Magnetic resonance imaging of the inner ear;8Phonoaudiological and psychological evaluation.The protocol for selecting patients was the following:a)Stable auditory thresholds within the past two years;b)Speech perception test – : < 40%;c)Prior experience with hearing aids;d)Sensorineural type dysacussThe main criteria for indicating the procedure were:Patients were selected based on the abovementioned criteria. All patients were informed about the indication for surgery, possible complications, objectives of therapy, and expected results. Patients were given a free informed consent form after detailed explanations.The institutional review board approved this study.-Age over 18 years.-Bilateral sensorineural dysacusis with little or no benefit from hearing aids.-Pure tone thresholds better than 65 dB at 125, 250, and 500 Hz, and worse than 85 dB at frequencies over 1,000 Hz.-Auditory discrimination for monosyllables below 40% at the best possible sound amplification.-Stable hearing loss in at least the past two years.Before surgery, conventional audiometry, speech perception tests with and without hearing aids, OAE (otoacoustic emissions) testing, BAEP , computed tomography and magnetic resonance imaging of the temporal bones were done in all patients. A psychological evaluation was carried out to discuss expectations about the implants.Implants were activated 45 days after surgery. Postoperative audiometry and speech perception tests were carried out.EASTM (21 mm) (The implant that was used in these cases was the Med-El MAnd FLEX (21 mm)  and 330. (21 mm) .Figure 2This project is ideal for insertion through the round window, a technique many otologic surgeons prefer because it is less traumatic. Premolded electrodes are more traumatic and are not easily inserted through the round window; they are therefore inadequate for preserving hearing. Many insertions were done through the scala vestibuli – rather than the scala tympani – with this type of electrodeth cranial nerve is monitored throughout the procedure. Electrodes are attached to the ipsilateral rami of the operated ear on the frontal, zygomatic, buccal, and mandibular areas; there is also a ground electrode attached onto the patient's thorax and a reference electrode  for the stimulatory pen  that is attached to the sternoclavicular area. We use a NIM-PulseTM .The 8Microscopy is essential and has revolutionized otologic procedures. We use a CARL ZEISS GMGH S88 Microscope™ with a camera and a digital video system for recording the procedures.th cranial nerve. The operative field is separated from the rest of the scalp by using microporeTM. Antibiotic prophylaxis is done with endovenous cefazolin (50 mg/kg) during induction of anesthesia.1The main landmarks are marked: tip of the mastoid, temporal line, retroauricular incision line, area of the internal component, and area of the microphone with the help of a mock-up ;Figure 42Antisepsis with 0.2% aqueous chlorhexidine, placement of sterile drapes and steri-drape™2 ;3Rectilinear retroauricular incision and dissection along anatomical planes. Preparation of a “cross” Palva flap  raising the four segments of the flap over the subperiosteal plane;4Removal of small fragments of fascia and temporal muscle to occlude the cochleostomy;5Simple mastoidectomy, identifying the lateral semicircular canal, the short ramus of the anvil, the posterior wall of the outer ear canal, the tegmen timpani, and the lateral sinus. Gathering a small amount of bone dust;6Thinning of the posterior wall of the outer ear canal, posterior tympanostomy, preservation of the incus buttress;7Preparation of the receiver well for the inner component of the intracochlear EAS on the squamous portion of the temporal bone (well) using a specific mock-up;8Irrigation of the cavity with povidone-iodine (10% povidone-iodine / 1% active iodine) for two minutes followed by abundant irrigation with lactated Ringer's solution™ ;Figure 59Irrigation of the cavity with ciprofloxacin (4mg/ml) for two minutes followed by irrigation with lactated Ringer's solution™ ;Figure 610Endovenous administration of dexamethasone (8 mg) before approaching the middle ear via a cochleostomy or through the round window;11Application of topical triamcinolone (40mg/ml) over the round window;12Opening the membrane of the round window; if this approach is impossible, the endosteous is opened by means of a cochleostomy;13Positioning the internal component on its well;14Preparation of the fascia graft; making a pinhole central orifice to allow the electrode to pass snugly to be placed in the cochleostomy/round window site;15Insertion of the electrode slowly and continuously during three minutes through the hole in the graft;16Positioning the muscle graft around the electrode to seal the cochleostomy. Placing bone dust to close the posterior tympanotomy;17Positioning the ground electrode under the muscle-periosteum flap;18™ 3.0 sutures on the Palva flap planes and subcutaneous tissue; skin closure with Nylon 4.0;Closure with Vicryl19Cleaning of the patient and placing an external compressive dressing;20Impedance testing, neural response telemetry (NRT), and a transorbital incidence radiograph are done to confirm the position of the intracochlear electrode.A patient is placed on horizontal dorsal decubitus with the head turned to the contralateral side. General anesthesia with orotracheal intubation is done. The operative field is prepared by ample trichotomy, antisepsis with 2% chlorhexidine, and placement of electrodes for monitoring the 8Note: it is worth noting that this surgical technique was not created or developed by our tem. We adopted this approach based on the experience that our team had acquired in courses, conferences, and reading the specific medical literature.Hearing rehabilitation through surgery (cochlear implant) with hearing preservation in patients with hearing remains in the low frequencies becomes a new treatment optionElectrodes on the lateral wall are less traumatic than premolded electrodes; they are preferable because of this feature and to preserve residual hearingTopical medication in the middle ear before placing an implant is a controversial topic; this concern, however, is justified. The purpose of using povidone-iodine and ciprofloxacin is to assure that the operative field remains sterile so that contamination of the inner ear is avoided as much as possible, thereby avoiding possible damage to the inner hair cells. The purpose of corticosteroids topically over the round window and systemically is to reduce intracochlear inflammation, thereby minimizing damage to the round window.There is a debate in the literature about the topical effect of endovenous drugs in the middle and inner earet al. have shown that the concentration of drugs in the cochlear perilymph is higher compared to the blood plasma at dosages for endovenous concentrations – such is the case of corticosteroids. These doses were measured in the cochlea and plasma based on equivalent endovenous dosagesBird The use of lactated Ringer's solution throughout the procedure, especially from the beginning of mastoidectomy, is justified because it is more similar to endolymph, which at least in theory reduces intracochlear trauma.The ossicular chain should be handled minimally; any disruption among ossicles should be avoided to preserve residual hearing; the ossicular chain can be compromised, which results in loss of conduction and reduced acoustic simulation. Any vibration of the ossicular chain may be conveyed to the inner ear and may cause cochlear injury, sensory loss, and further loss of residual hearingSeveral authors consider the approach to electrode insertion – cochleostomy or through the round window – as one of the most important steps to reduce intracochlear damageTherefore, insertion of the electrode through a cochleostomy anterior and inferior to or directly through the round window is currently the method of choice; this approach increases the certainty of accessing the scala tympani and has a lower potential for cochlear traumaElectrodes should be inserted slowly, continuously, and at a constant rate. Rapid electrode insertion may cause fluid-mediated injury. Care should be taken not to introduce contaminants  into the intracochlear compartment, which would increase local inflammation. Electrodes should fit snugly, but without resistance; care should be taken not to suction endolymph. There is an exponential increase in resistance to progression after the electrode is inserted by about 15 mm. The electrode specifications, its material (it should be as inert as possible to minimize foreign body inflammation), flexibility, characteristics of the internal arrays, diameter, length, and others, are also important factorsThe cochleostomy is sealed with a temporal fascia graft, which should not be too large so as not to affect the mobility of the ossicular chain and to avoid inflammation from altering the middle ear homeostasisThe steps of surgery were planned to preserve residual hearing and to reduce cochlear injury as much as possible.Although much has been said about EAS, this mode of therapy is rarely undertaken in Brazil. Our unit has pioneered this procedure in this setting, and not many such procedures have been done. We point out that cochlear manipulation surgery aiming to preserve hearing is difficult.We described the technique for EAS implants as applied to the cases operated at our unit. This technique differs from conventional cochlear implant routine by including steps aiming at hearing preservation. The technique is complex and has nuances that make it difficult to carry out adequately.We believe that cochlear implants with hearing preservation opens a new era in otology and the rehabilitation of patients with hearing loss.