During laser treatment a darkening of the TCs lumina was achieved in 91.3% of lesions. All these lesions fully resolved, whereas TCs, which showed no darkening of their lumen in OCT persisted and required re-treatment with laser. Additional anti-VEGF injections were indicated in only one eye (8.3%). The darkening of the TCs lumina visible in OCT might provide an image-biomarker that indicates successful coagulation of aneurysmatic lesions. Consequently, a significant functional and morphological improvement with need for anti-VEGF treatment in only one eye, was achieved.In this retrospective study the morphological response of teleangiectatic capillaries (TCs) to focal laser treatment and the functional and morphological outcome after Indocyanine green angiography (ICGA)-guided laser therapy was evaluated. TCs in eyes with diabetic macular edema (DME) were treated with laser therapy. The immediate and subsequent reaction of the TCs lumina to direct photocoagulation was monitored with customized OCT single scans. Additionally, patients were treated with intravitreal anti-VEGF as needed. 12 eyes of 9 patients with treatment naive (6 eyes) and pretreated (6 eyes) DME were followed-up for a mean of 24 months (± 8.1SD). Best-corrected visual acuity improved from 0.25 logMar (± 0.2SD) to 0.12 (± 0.10SD; Information concerning the registration of the trial: date of registration: 11th of december, 2019. Trial registration number: 107/2019. For many years fluorescein angiography (FA)-based focal/grid macular laser has been the go-to treatment for patients with DME4.Vision loss due to diabetic macular edema (DME) and its treatment have become a major socioeconomic burden8. The potential risks of laser therapy, such as the development of a choroidal neovascularization, foveal burns or enlargement of laser scars in the absence of an additional functional benefit seem to have underpinned the paradigm of “anti-VEGF injections alone”9.However, since the introduction of intravitreal anti-vascular endothelial growth factor (VEGF) therapy, macular laser has lost its role as a primary treatment in patients with center-involving DME. Data from randomized clinical trials examining fluorescein angiography-based central laser therapy (e.g. modified ETDRS laser treatment) in addition to intravitreal anti-VEGF showed no statistically significant benefit compared with intravitreal anti-VEGF monotherapy for visual acuity outcomes19.With the introduction of OCT imaging non-invasive visualization of intra- and subretinal fluid became possible, thereby greatly impacting the diagnosis of diabetic macular edema and other retinal disease23. It was found that, compared to FA, ICGA depicts microaneurysms more specifically in the area of edema, especially in the late-phase of the angiography21. In the early phase of the angiogram ICG starts to partially accumulate in specific aneurysms until they become completely filled in the later phases (≥ 10 min). When the dye is washed out of the retinal vessels and other microaneurysms, some lesions show prolonged ICG staining23.However, established standards for focal laser therapy are still based on FA findings and do not include guidance by OCT imaging for the detection of potential laser targets. Furthermore, an increasing amount of recently published data highlights the superiority of indocyanine green angiography (ICGA) compared to fluorescein angiography (FA) for detecting specific microaneurysms that are responsible for the formation of DME21. The presence of TCs has also been described in patients with macular edema secondary to retinal vein occlusion and idiopathic macular teleangiectasia26. There is however very little data on the functional and morphological outcome of ICGA-guided laser therapy of TCs in patients with diabetic macular edema20. Our study presents long-term results of an ICGA-guided approach to focal laser therapy. Additionally, we used customized OCT single-scans to evaluate the immediate as well as the consecutive morphological response of the TCs to their direct laser photocoagulation, for which the term “OCT monitoring” will be used in this manuscript.Because of this distinct staining-pattern it was suggested to term these lesions “teleangiectatic capillaries” (TC) to differentiate them from common microaneurysmsIn this retrospective study we evaluated data from 12 eyes of 9 patients with center-involving DME  > 300 µm) in whom TCs were selectively targeted with focal laser photocoagulation in the macular region between October 1, 2014 and August 22, 2018. This study was approved by the Institutional Review Board Burgenland . Due to the retrospective nature of the study the Institutional Review Board Burgenland  has waived off the need for informed consent. The study adhered to the tenets of the Declaration of Helsinki. All laser treatments were performed by the same retina expert (PD) in an ophthalmological practice.In patients with center-involving DME, FA and ICGA were simultaneously performed to enable measurements to be transferred from ICGA to FA images and vice versa Figs. A, B, 4A.FA and ICGA images of the patients were displayed on a monitor directly next to the laser device to facilitate localization of the TCs during the procedure. Laser treatment was performed using a laser system operating at a wavelength of 577 nm  with the following settings: impulse duration 20 ms, spot size 60–100 µm, 100–350 mW, central mirror of Goldmann contact lens. Methocel 2% contact gel was administered prior to insertion of the contact lens. Laser bursts were applied directly on the surface of the TCs until a minimal greyish-white discoloration of these lesions was seen. After this initial treatment-phase the customized OCT scans, placed directly through the TCs, were used to monitor changes in the reflectivity of their lumina. As soon as a darkening of a TCs lumen was seen in the OCT single scans no further laser bursts were applied to this lesion . The repeated acquisition of the customized OCT single scans during laser treatment was usually performed one to three times until a darkening of the TCs lumina was visible. In the ophthalmological practice where all laser treatments were performed, the laser machine is positioned in a 90° angle in relation to the OCT device. Thus, the patient can remain seated by turning 90° to the side, which facilitated time efficient acquisition of the repeated OCT single scans during laser treatment.BCVA was assessed using 6 m Snellen visual acuity charts. OCT was routinely performed at each visit during the follow-up period. CST was measured in the central field of the macular ETDRS grid Fig. G–J. Multp-value  were calculated. Statistical analyses were calculated with SAS 9.4 and R 4.0.5. The significance level was set to alpha = 0.05.The comparison of BCVA and CST values between baseline and month 6–8 as well as month 12–15 and the last visit before November 30, 2019 were performed by mixed models (SAS Proc mixed). For statistical analysis BCVA data was converted from Snellen VA to the logarithm of the minimum angle of resolution (logMAR), The number of degrees of freedom was calculated using the Kenward–Roger-approximation. The difference to baseline was the dependent variable. Patient was taken as a random factor. For analysis of month 6–8 and month 12–15, respectively, only the intercept was included in the model, for analysis of the last visit, the follow-up time (centered at a mean follow-up time of 23.75 months) was additionally included. An estimate for the mean difference to baseline with 95% confidence limits and the We calculated the average duration until shrinking and resorption of the TCs was seen during follow-up. Further, two graders (F.D and A.D) independently evaluated the reflectivity of the TCs lumina in the OCT single scans that were acquired before and during laser therapy. Reflectivity of the lumen was graded as hypo- or hyperreflective, compared to the surrounding vessel wall Figs. F, G or aAdditionally, we measured the distance from the maximum height of the edema at baseline to the largest TC in µm and we determined the mean number of laser targets treated per eye, the mean distance from the fovea to the laser target closest to the fovea, the mean size of the TCs treated with laser and the number of retreatments with laser as well as the proportion of patients requiring intravitreal injections of anti-VEGF in addition to laser therapy.The mean age of the patients  was 71 years (± 9.6 SD), ranging from 61 to 84. The mean follow-up was 24.0 months (± 8.1 SD), ranging from 11 to 43 months. Prior to ICGA-guided laser photocoagulation 6 of the 12 eyes were pretreated with anti-VEGF. 4 of the 6 pretreated eyes additionally had FA-based focal/grid laser photocoagulation prior to anti-VEGF therapy and the other 2 pretreated eyes received anti-VEGF only. 6 eyes were treatment naive. In 5 patients (8 eyes) HbA1c values were available before laser therapy as well as during the follow up. Mean HbA1c before laser therapy was 6.8% ranging from 6.3 to 7.0 and 7.01% after laser therapy ranging from 6.4 to 7.2.p = 0.05, mixed model), from baseline to month 12–15 (p = 0.02) as well as a tendency towards statistical significance from baseline to each last visit of the patients (p = 0.06). CST at baseline was 418 µm  and decreased to 349 µm  after a mean of 1.3 months after laser therapy. Mean CST further decreased statistically significantly to 311 µm  at months 6–8  and remained stable at 299 µm  at months 12–15 (p = 0.001) and at 298 µm  at the last visit (p = 0.001). Morphological and functional results for each patient are presented in Table The mean BCVA at baseline was 0.25 logMar  and improved to 0.19 logMar  at months 6–8 and to 0.13 logMar  at months 12–15. BCVA at the patients last visit was 0.12 . BCVA improvements were statistically significant from baseline to month 6–8 , ranging from 1 to 3. The mean distance from the largest TC to the maximum height of the edema was 413 µm (± 218 SD) ranging from 51 to 743 µm. The mean size of the TCs was 170 µm . The mean distance of the TCs closest to the fovea was 960 µm , ranging from 549 to 2039 µm. According to the analysis of both graders prior to laser therapy all TC’s presented with a hyporeflective lumen compared to the surrounding vessel wall in the OCT single scans . During the first laser treatment an immediate darkening of the TCs lumina could be monitored with OCT in 10 out of 12 eyes , a recent advancement in ophthalmic imaging, allows non-invasive detection of blood flow in and around the macular region. Its utility to detect TCs has been evaluated in a series of 20 patients undergoing both ICGA and OCTA examination. It was found that TCs showed rather poor visibility in OCTA, possibly due to presence of intraluminal material inside the TCs, that reduces the flow velocity below the threshold of the OCTA device so that they are no longer detectable33. While resolution of ultra-widefield imaging devices is useful to detect peripheral ischemia and neovascularization and still might be sufficient to depict TCs, we know from clinical experience, that small retinal vessels in the macular region are commonly not well visualized. This makes the exact localization of the TCs more difficult, since those small retinal vessels serve as reference points to locate the TCs during laser therapy. This is particularly important in patients with a lot of microaneuryms but only few TCs. What is more, TCs that are involved in the formation of center involving DME are rarely located outside the 30° field of view imaging which can be performed with the Spectralis Heidelberg HRA + OCT device21. Additionally the scan planning tool available for this device allowed us to create the OCT single scans that we used to monitor the darkening of the TCs lumina.Ultra-widefield imaging, another innovation in retinal imaging, allows for a standardized evaluation of diabetic retinal changes up until the far peripheryIn times of emerging automation and navigated laser systems, implementing OCT monitoring in such devices to enable the immediate evaluation of the treatment response automatically would be of great interest to further optimize the outcome, safety and accuracy of focal laser therapies in patients with DME.The automated real-time tracking function of the OCT device, which enables up to 100 OCT scans, acquired at the exact same location, to be averaged, was crucial for obtaining images of sufficient quality to assess changes in the reflectivity of the TCs lumina. Thus, intraprocedural monitoring of the treatment response was possible, despite the use of a contact agent, as required for focal laser therapy with a contact lens. Combining OCT monitoring of the treatment response with an OCT scan planning tool software allowed the post hoc customization of OCT scans, that are based on the patient’s ICGA images, any time after completion of the ICGA examination. Thus, the operator of the device does not necessarily have to acquire OCT scans localized exactly at the spots of interest during the ICG examination, which is often challenging in routine clinical practice. Post hoc scan planning further reduces the number of scans needed to depict all potential laser targets, because at least two lesions can be imaged with a single OCT scan. This allows for even more time efficient intraprocedural monitoring of the treatment response, making it feasible for clinical practice.20. They applied focal laser treatment to TCs larger than 150 µm in eyes with persistent DME after treatment with anti-VEGF. However, their patients differed from ours in terms of baseline BCVA. The mean BCVA at baseline was 20/32 Snellen VA in our patients and 20/200 in theirs. In their study, BCVA improved in 2 eyes and remained stable in another 2 eyes, while all eyes showed morphological improvement without additional therapy during a follow-up of 6 months. In our patients BCVA improved in 8 eyes, remained stable in 3 eyes and worsened only in 1 eye. Morphological improvement was achieved in all of our patients. In their patients mean CST at baseline was 515 µm and the mean number of TCs was 3.420. In our patients mean CST at baseline was 418 µm and the mean number of TCs was 1.5. These differences suggest a connection between more severe DME and a higher number of TCs per eye.Our laser protocol is most similar to that of a pilot study published by Paques et al., in which four eyes of three patients with DME were followed up for 6 months after ICGA-guided laser therapy of TCs22. However, despite a mean of 22 laser targets per eye, compared with 1.5 targets in our patients, the reduction in CST they achieved was similar to the results of our study. This suggests, that there are certain ICG-positive aneurysms, probably TCs, that are more relevant to edema formation than others22. What’s more, 63% of their patients required retreatment with laser after 3 months, compared with 17% in our patients within the first year22. This indicates that funduscopic evaluation of the lesion closure, as available in current automatically navigated laser devices, could be misleading. Further, careful selection of specific, possibly larger lesions, that show staining in the late phase (> 10 min) of the ICGA, instead of targeting all lesions visible in earlier phases of the angiography, could lead to a similar functional and morphological outcome. Aneurysm size may be one factor responsible for the amount of exudation, as laser therapy showed only a modest effect in clinical trials that mainly included aneurysms of 150 µm or smaller34. Apart from the lesion size, late ICGA staining seems to distinguish highly exudative lesions from others24. Nevertheless, other contributing factors, such as the wall thickness or the presence of intraluminal material to the amount of exudation caused by the individual lesion should be investigated in more detail to distinguish highly exudative ICG-positive lesions from others, and to optimize the treatment effect while minimizing the damage inflicted to the retina.Another approach highlighted the functional and morphological benefit of ICGA-guided photocoagulation of microaneurysms, but not TCs in particular, using an automated navigated laser photocoagulation system  in 8 eyes of 6 patients with DME pretreated with anti-VEGF35. Fluorescein, which is hydrophilic, would be blocked by such intraluminal material. Interestingly, some of these ICG-positive lesions are not detectable or become only partially filled in FA due to blockage of fluorescein by some intraluminal material. These incompletely filled aneurysms are best visualized in the early phase of FA36. Adaptive optics scanning light ophthalmoscope (AOSLO) fluorescein angiography combined with AOSLO reflectance images confirmed that partial filling of aneurysms is seen due to the presence of intraluminal material37. What is more, some TCs are not visible in early- and late-phase FA and might therefore easily be overlooked without additional ICGA (Fig. The reason why ICGA is able to depict lesions that are responsible for edema formation, might lie in its chemical properties. It is hypothesized based on histology studies that ICG, because of its amphiphilic properties, binds to hydrophobic intraluminal material such as fibrin, lipids or red blood cell components, which are suggestive of chronic leakage out of these lesionsThe limitations of our study are its retrospective nature and the relatively low number of patients. However, to the best of our knowledge, this is the largest study with a long follow-up time on this topic to date.38.Depiction of TCs with ICGA, combined with the possibility to systematically monitor the immediate treatment response of laser targets during laser treatment with OCT, as shown in our study, encourages a re-evaluation of the role of focal laser therapy in patients with center-involving DME. Additionally, there is some evidence that the presence of late-phase ICG positive aneurysms, but not FA positive aneurysms, is associated with recurrent DME under anti-VEGF therapyHowever, there is still need for further studies investigating the connection between the anti-VEGF treatment response and the presence of TCs. A large clinical trial comparing the outcome of intravitreal injections of anti-VEGF with and without additional ICGA-targeted laser, including OCT monitoring of the immediate treatment response, would be of utmost importance to better understand the role of ICG-guided central macular laser for the treatment of patients with center-involving DME.


Cellular senescence is a process that leads to a state of irreversible cell growth arrest induced by a variety of intrinsic and extrinsic stresses. Senescent cells (SnCs) accumulate with age and have been implicated in various age-related diseases in part via expressing the senescence-associated secretory phenotype. Elimination of SnCs has the potential to delay aging, treat age-related diseases and extend healthspan. However, once cells becoming senescent, they are more resistant to apoptotic stimuli. Senolytics can selectively eliminate SnCs by targeting the SnC anti-apoptotic pathways (SCAPs). They have been developed as a novel pharmacological strategy to treat various age-related diseases. However, the heterogeneity of the SnCs indicates that SnCs depend on different proteins or pathways for their survival. Thus, a better understanding of the underlying mechanisms for apoptotic resistance of SnCs will provide new molecular targets for the development of cell-specific or broad-spectrum therapeutics to clear SnCs. In this review, we discussed the latest research progresses and challenge in senolytic development, described the significance of regulation of senescence and apoptosis in aging, and systematically summarized the SCAPs involved in the apoptotic resistance in SnCs. Aging is a time-dependent functional decline that affects most living organisms. It is characterized by a progressive loss of physiological integrity, causing impaired function and increased vulnerability to death . Aging rin vitro  . Howeverin vitro and in vivo  when they became senescent . Howeverp53 is a well-known tumor suppressor that acts as a double-edged sword in the regulation of cellular senescence, aging, and tumor . The hosin vivo  are evolutionarily conserved proteins whose expression is induced by different kinds of stresses . They arAmong the HSP families, HSP90 has been implicated as an anti-apoptotic and pro-survival factor in SnCs, and the inhibitor of HSP90 was identified as a new class of senolytic , 2018. ITheoretically, the autophagy-enhanced cellular immune responses may facilitate the immune system to recognize and eliminate SnCs, which has yet to be validated. The relationship between autophagy and cellular senescence is inconclusive  is a transcription factor complex consisting of homo- and heterodimers of five members of the Rel family including RelA (p65), RelB, c-Rel, NF-κB1 (p50/p105), and NF-κB2 (p52/p100). The NF-κB pathway transcriptionally controls a large set of target genes that play important roles in cell survival, inflammation, and immune responses . PreviouThe mitogen-activated protein kinase (MAPK) family can act as an upstream of the NF-κB pathway. MAP kinases are grouped into three families: stress-activated protein kinases (p38/SAPKs), Jun amino-terminal kinases (JNKs), and extracellular-signal-regulated kinases (ERKs). In cellular senescence, p38 induces the expression of the SASP largely by increasing NF-κB transcriptional activity . JNKs plThe insulin/IGF signaling pathway plays a major role in determining the rate of aging in many species , which iCaspases are a family of endoproteases that provide critical links in cell regulatory networks controlling inflammation and cell death. They are inactive until being cleaved at specific aspartate residues . Wang’s Survivin is an important anti-apoptotic protein. It is highly expressed in most tumors, which is also generally arised in cells of older individuals . Al-Khal2+-dependent actin regulatory protein, were increased in senescent HDF. Downregulation of gelsolin in senescent HDF resulted in increased sensitivity to menadione-induced apoptotic cell death  on the cell surface . The bes+ T cells to inhibit immune responses against SnCs. Accordingly, blocking the interaction between HLA-E and NKG2A boosts immune responses against SnCs  were developed to selectively target SnCs and have shown great potential to delay aging and to treat age-related diseases. It is encouraging to know that several senolytics have been approved for clinical trial and have shown beneficial effects . The lat


Cochlear implants (CIs) have been remarkably successful at restoring hearing in severely-to-profoundly hearing-impaired individuals. However, users often struggle to deconstruct complex auditory scenes with multiple simultaneous sounds, which can result in reduced music enjoyment and impaired speech understanding in background noise. Hearing aid users often have similar issues, though these are typically less acute. Several recent studies have shown that haptic stimulation can enhance CI listening by giving access to sound features that are poorly transmitted through the electrical CI signal. This “electro-haptic stimulation” improves melody recognition and pitch discrimination, as well as speech-in-noise performance and sound localization. The success of this approach suggests it could also enhance auditory perception in hearing-aid users and other hearing-impaired listeners. This review focuses on the use of haptic stimulation to enhance music perception in hearing-impaired listeners. Music is prevalent throughout everyday life, being critical to media such as film and video games, and often being central to events such as weddings and funerals. It represents the biggest challenge for signal processing, as it is typically an extremely complex acoustic signal, containing multiple simultaneous harmonic and inharmonic sounds. Signal-processing approaches developed for enhancing music perception could therefore have significant utility for other key issues faced by hearing-impaired listeners, such as understanding speech in noisy environments. This review first discusses the limits of music perception in hearing-impaired listeners and the limits of the tactile system. It then discusses the evidence around integration of audio and haptic stimulation in the brain. Next, the features, suitability, and success of current haptic devices for enhancing music perception are reviewed, as well as the signal-processing approaches that could be deployed in future haptic devices. Finally, the cutting-edge technologies that could be exploited for enhancing music perception with haptics are discussed. These include the latest micro motor and driver technology, low-power wireless technology, machine learning, big data, and cloud computing. New approaches for enhancing music perception in hearing-impaired listeners could substantially improve quality of life. Furthermore, effective haptic techniques for providing complex sound information could offer a non-invasive, affordable means for enhancing listening more broadly in hearing-impaired individuals. Cochlear implants (CIs) recover hearing for severely-to-profoundly hearing-impaired individuals by electrically stimulating the cochlea. They deploy an array of up to 22 microelectrodes, replacing the approximately 3,500 hair cells that transfer sound to the brain in normal-hearing listeners. Despite the fact that only limited sound information can be provided through this small number electrodes, CIs have been remarkably successful at recovering access to speech in quiet listening conditions . HoweverSeveral studies have recently shown that haptic stimulation can enhance CI listening by allowing access to sound features that are poorly transferred through electrical CI stimulation see . This “eThis review will focus on the use of haptic stimulation to enhance music perception in hearing-impaired listeners. Most people in the deaf community report being involved in music activities  and musiWhen considering whether a haptic system might enhance music perception in hearing-impaired listeners, it is important to first establish the limits of music listening when hearing is impaired. It has been reported that, after a CI is implanted, only around 15% of adults enjoy listening to music  and arouNumerous studies have explored which of the auditory features within musical pieces can be effectively extracted by hearing-assistive device users. CI users typically perform well at basic rhythm , tempo , and metHA users have similar spectral and temporal modulation thresholds to normal-hearing listeners  and, likVision plays an important role in music perception for hearing-impaired listeners. Viewing the performer and reading lyrics can increase their musical enjoyment  and raveSeveral other factors are known to have important influences on music perception for hearing-impaired listeners. For example, the age at which hearing impairment occurred, the amount of residual hearing retained for CI users, and the efficiency of sequential cognitive processing are predictive of pitch and timbre perception . Age is To establish how haptic stimulation might effectively augment listening, this section compares the sensitivity of the tactile system to the impaired auditory system. First, sensitivity to frequency, intensity, and temporal features will be considered , rather than spatial location . CriticaIn addition to devices developed to augment music perception, several devices have been developed to aid those with sensory impairments by substituting one sense with another. An early example of a sensory substitution device is the Teletactor, developed in the 1920s, which transferred sound to deaf listeners through tactile stimulation on the hand , 1926. TSome of the key design considerations when developing a modern haptic device for enhancing listening are discussed by Music is commonly accessed through streaming services. This opens the possibility of using signal-processing approaches that cannot be applied in real-time or that are non-causal (require the ability to look ahead). It also opens the possibility of using pre-trained machine-learning algorithms that are selected between based on metadata sent through the streaming service. These algorithms could be trained using the numerous high-quality musical corpora available, which can be supplemented using advanced automated music generation algorithms . So-callIt is important to first establish the goal when converting audio to haptics for music enhancement. One approach is to remove elements that reduce clarity when audio is transferred at a low-resolution . One example of this is spectral complexity reduction, in which the frequency spectrum is sparsened and simplified, using methods such as principal component analysis . SpectraSome basic methods for separating sound sources have already been used for converting audio to haptic stimulation. One haptic signal-processing approach uses an expander, which amplifies loud sounds, to extract speech from background noise when the signal-to-noise ratio (SNR) is positive  and the HearingFitness program , which use big data collected from devices in the real world to inform policy-making . In futuTo implement a remote data processing pipeline of this sort, exploitation of cutting-edge technology and further research are required. It should be noted that, in practice, simpler systems that collect user feedback to optimize new iterations of algorithms might be developed before a full pipeline like that proposed is implemented. One key technology for the proposed pipeline is wireless data streaming. This can be achieved using the latest Bluetooth Low Energy technology, which allows multiple simultaneous data streams, has low power usage, and is already integrated into many of the latest hearing-assistive devices. Another critical element is the development of a smartphone app for collecting user feedback, which must have a high level of data security and privacy. User feedback is likely to be important as music perception varies substantially across hearing-impaired listeners due to factors such as previous musical experience . The appIn addition to user- and stimulus-based optimization of signal processing, steps should be taken to ensure that haptic stimulation is perceived as uniformly as possible across users. One simple way to do this is to adjust the stimulation intensity based on each user’s detection thresholds (as is done for hearing-assistive devices). It may also be important to adapt the intensity based on the fitting of the device on the body. The fitting  can substantially alter the amount of pressure applied to the haptic motor and the coupling with the skin. Techniques have recently been developed to estimate the pressing force on the motor and dynamically calibrate it . Such teMusic perception is often significantly impaired in those with hearing loss. Critical factors are the loss of ability to discriminate sounds of different frequencies and a reduction in dynamic range. Recently, it has been shown that haptic devices can be highly effective at providing intensity , 2021a,bSeveral critical technologies have been developed in recent years, which can be exploited in future haptic devices. These allow faithful haptic signal reproduction, advanced signal processing, wireless communication between hardware components , long battery lives, and rapid prototyping and manufacturing. These technologies give scope for vast improvements to current haptic devices for enhancing hearing. In addition, several key emerging technologies and methods have been identified, which further expand the potential for haptic enhancement of music perception. These include cloud computing and cutting-edge machine-learning approaches. Exploitation of these new technologies could considerably increase haptic enhancement of listening and allow a dramatic expansion in access to music and other media for hearing-impaired listeners.Another consideration raised in this review is the interaction between haptic, audio, and visual stimulation. It was highlighted that significant sound information from music is accessible through vision, particularly pitch interval size and direction. Future work should establish whether critical sound information, such as pitch, provided through haptic, audio, and visual modalities can be effectively combined to enhance discrimination. It will also be critical to explore how providing sound information through non-auditory senses can alter auditory perception. This could determine whether future research on haptic enhancement aims to restore conventional music perception or whether it instead seeks to offer an alternative way to experience music.In addition to enhancing music listening, there is significant potential for haptics to be used for enhancing musical performance in hearing-impaired individuals. Of particular interest might be enhancement of vocal performance. CI users often have considerable difficulties when singing, particularly in producing the correct pitch . There hThis review has discussed the enormous potential of haptic stimulation to enhance music listening. It is estimated that around 1.6 billion people across the world have hearing loss, with this number expected to increase rapidly . AlongsiIf effective and accessible systems are developed, as well as directly enhancing music enjoyment, they could substantially improve access to and enjoyment of media (such as films and documentaries), video games, and social events, such as weddings. Furthermore, given that music is an extremely challenging signal because of its complexity, progress in this area could have substantial benefits for enhancing communication and spatial awareness in complex everyday acoustic environments. Thanks to inexpensive core technologies, haptic devices could become widely accessible, including in low- and middle-income countries, and bring substantial improvements in quality of life for those with hearing impairment.The author confirms being the sole contributor of this work and has approved it for publication.The author declares that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest.All claims expressed in this article are solely those of the authors and do not necessarily represent those of their affiliated organizations, or those of the publisher, the editors and the reviewers. Any product that may be evaluated in this article, or claim that may be made by its manufacturer, is not guaranteed or endorsed by the publisher.


Studies suggest that enzyme-replacement therapy (ERT) is crucial to the survival of patients with infantile-onset Pompe disease (IOPD). Hearing impairment (HI) is one of the clinical sequelae observed in long-term survivors. However, the benefits of early ERT for hearing outcomes have not yet been reported. This study aimed to investigate the impact of early ERT on IOPD patients.This retrospective longitudinal study recruited IOPD patients who were referred by newborn screening for confirmatory diagnosis based on our rapid diagnostic criteria and received early ERT treatment between January 1, 2010, and January 31, 2018. The hearing test battery included a tympanogram, otoacoustic emission, auditory brainstem evoked response (ABR), pure-tone audiometry or conditioned play audiometry.Nineteen patients with IOPD were identified, 6 of whom had hearing impairment (HI); 1 had conductive HI, 2 had sensorineural HI  and 1 had moderate mixed-type HI. Two patients failed the newborn screening test and had mild HI in the ABR. The mean age of the initial time to ERT was 11.05 ± 4.31 days, and the HI rate was 31.6% (6/19).Our study is the largest cohort to show the characteristic hearing outcomes of IOPD patients after ERT. Early ERT within 2 weeks after birth may contribute to better hearing outcomes. Clinicians should be vigilant in testing for the hearing issues associated with IOPD and should intervene early if any HI is detected. Pompe disease, which is also known as glycogen storage disorder type II or acid maltase deficiency, is an autosomal recessive lysosomal storage disorder that is caused by a deficiency of the lysosomal enzyme acid alpha-glucosidase (GAA) –4. The iPompe disease leads to the progressive accumulation of glycogen in specific organs and tissues, especially muscular  and nervous tissues, causing abnormal motor and neural functions, including hearing loss. The presentation of glycogen storage in the organ of Corti in a knockout mouse model of Pompe disease, including in the inner and outer hair cells, the supporting cells, the stria vascularis, and the spiral ganglion neurons, suggests that this hearing impairment may be related to a cochlear pathology , 12.This disease shows an extremely wide spectrum of symptom severity and is usually classified into three categories according to the time of onset: infantile-onset Pompe disease (IOPD) and late-onset Pompe disease (LOPD), and in between, non-classical infantile-onset Pompe disease , 14. PatEarly enzyme-replacement therapy (ERT) with recombinant human alpha-glucosidase can improve the survival rate, reverse cardiomyopathy, and improve motor function in patients with Pompe disease –19. AlthAs in other lysosomal storage disorders, while there is a consensus in favor of early ERT before the occurrence of irreversible damage when treating IOPD , our preIn Taiwan, Pompe disease has been included in the nationwide newborn screening program since 2008. This study included children who were referred to Taipei Veterans General Hospital (TVGH) and received a confirmed diagnosis of IOPD between January 1, 2010 and January 31, 2018. After 2010, all the referred newborns were given ERT starting within 4 h of admission if they had the following manifestations: (1) general hypotonia; (2) an elevated creatine kinase level > 250 m/L); (3) extremely low GAA activity upon initial dried blood spot analysis (< 0.50 mmol/L/h); and (4) a high left ventricular mass index (> 80 g/m2) , 25; con0 m/L; (3Newborn hearing screening was performed by automatic auditory brainstem evoked response (aABR) after birth. Behavioral test techniques, such as conditioned play audiometry (CPA), were applied for patients under 5 years old. Pure‐tone threshold testing was performed for both air conduction and bone conduction. The average air conduction thresholds at frequencies of 500, 1,000, and 2,000 Hz were used to classify the type of hearing loss (the conductive component in case of an air‐bone gap of ≥ 15 dB) and to grade the amount of hearing loss according to the American Speech-Language-Hearing Association (ASHA) definition of the degree of hearing loss . The degThe quantitative data were summarized as the means ± standard deviation (SD), and the categorical variables were summarized as percentages. All the statistical analyses were performed using IBM SPSS 20.0 software .Nineteen patients with IOPD were included in the current study. Table Table In this study, we reported the hearing test results of the IOPD patients, and we found that the patients in our cohort had early ERT initiation time and a lower hearing impairment rate . To our knowledge, this is the largest cohort of patients to be investigated for the audiological characteristics of IOPD after receiving early ERT.Hearing loss in IOPD may be sensorineural, conductive, or a mixed type. In the present study, sensorineural hearing loss is the most common type, which is consistent with earlier studies , 22. MarConductive hearing loss can also be observed in patients with Pompe disease , 21, 31.The natural course of IOPD without ERT would be progressive cardiomyopathy and generalized myopathy or muscular hypotonia . FurtherAlthough impaired hearing function has been reported in patients with IOPD, many studies mentioned neither the test battery used for the hearing evaluations nor the criteria used to report the degree of hearing loss, thus making it difficult to compare the results among different groups Table . Hahn etIt is difficult to compare our outcomes with those of other groups due to a lack of detailed hearing data from their studies. Several reasons may underlie the better hearing outcomes obtained in the current study. The racial difference should be considered first. The cohorts of both Chien et al. and our study are from Asian populations , and theOur study has several limitations. First, since Pompe disease is rare, a small sample size is an inevitable issue. In addition, ERT was first approved for Pompe disease treatment in Taiwan in 2006, and all the follow-up instances have taken place since then, limiting the length of the follow-up period to date. However, our study is one of the largest cohorts to provide data on both early ERT treatment and hearing examination results. Third, there is a paucity of direct evidence to show whether better hearing results after ERT are due to reduced glycogen accumulation in the auditory system. In fact, the blood-labyrinth barrier, which is similar to the blood–brain barrier, may limit the effects of the recombinant enzymes used for ERT –38. FurtOur study is the largest cohort to provide detailed data of hearing outcomes in IOPD patients who received early ERT treatment. Early diagnosis and early initiation of ERT within two weeks after birth appear to contribute to improved hearing outcomes for patients with IOPD. Clinicians should be vigilant for the hearing issues associated with IOPD and intervene early if any hearing impairment occurs in their IOPD patients.


Older adults with hearing loss often report higher fatigue due to effortful listening. We evaluated whether self-reported hearing ability is associated with perceived physical and mental fatigability (a more sensitive measure than fatigue) using the Pittsburgh Fatigability Scale (PFS). Older adults  from the Long Life Family Study Visit 2  completed PFS and self-reported hearing ability . Age-adjusted PFS Physical and Mental scores were 2.3 and 2.5 lower, respectively, for worse vs. better hearing (p<.0001). Generalized estimating equations adjusted for family-relatedness, site, age, sex, cognitive function , education, and self-reported health. Compared to individuals with better hearing, those with worse hearing had a 42% and 44% greater odds of physical (≥15)  and mental(≥13)  fatigability, respectively. These observed associations may potentially be explained via complex psychosocial and cognitive aging pathways (e.g. effortful listening) to be examined in future work.


Telocytes (TCs) are a new type of mesenchymal cells that have been discovered recently in many organs and tissues. However, studies of TCs in the avian reproductive system are still at the beginning. Chickens are one of the world's most popular domesticated animals, providing inexpensive but valuable proteins and nutrients from chickens and eggs to nourish the human bodies. Chickens have important scientific value; thus, understanding the reproductive system regulations seems to be important. The utero-vaginal junction is involved in the regulation of sperm storage. The sperm storage tube (SST) in the utero-vaginal junction stores sperm. The purpose of this study was to investigate the existence of TCs in the utero-vaginal junction of the chicken, and their structural relationships with the sperm storage tube and surrounding cell types. We studied the morphology, ultrastructure, and immune characterization of TCs.n = 10) were used for Masson's staining, fluorescent in situ hybridization technique (FISH), and transmission electron microscopy (TEM) analysis. The results showed that TCs were present in the utero-vaginal junction. TCs appear as CD34 immunopositive and C-kit immunopositive. They were identified especially via small-body and long-protrusion telopodes (Tps) containing Podomers (Pm) and Podoms (Pd). The Tps were bent, folded, and intertwined with each other, sometimes in the shape of a labyrinth. The Tps were embedded between collagen fiber bundles, smooth muscle bundles, and around blood vessels and releasing vesicles. TCs surround these glands, forming heteromorphic cell connections with surrounding lymphocytes and plasma cells, smooth muscle cells, blood vessels, collagen fibers, and fibroblast-formed homotypic or allotypic connections in a complex three-dimensional network structure. This study provides a morphological basis for the possible role of TCs in regulating the utero-vaginal junction physiological role and in intercellular communication.The utero-vaginal junction of 4-month-old healthy adult chickens ( Telocytes (TCs) are a new type of mesenchymal cells named by Romanian Professor Popescu and Italian Professor Faussone in 2010. This cell type was first discovered in the human pancreas by Popescu and his team in 2005 because its form is similar to the interstitial cells of Cajal (ICCs) in the digestive tract. He speculated that these cells may be a form of ICC, called simple Cajal interstitial cells  .2+ channel, and estrogen and progesterone receptors . In contrast, the fowl oviduct is a unilateral organ, coiling in the abdominal cavity on the left side, and composed of the infundibulum, magnum, isthmus, uterine, and vagina. The Japanese scholar Fujii reported in 1963 that the oviduct and uterus and the integration of the vaginal mucosa lamina propria gland can store sperm . The oviin situ hybridization. Tissue samples were dehydrated in a series of graded concentration ethanol  . Tissue samples were first embedded and made into paraffin wax blocks, and then tissue sections were cut into 5 μm. Sample preparation was conducted according to accepted international standards.Ten healthy adult laying chickens, 4 months of age, weighing 2.0–2.5 kg per chicken were used in this study. The feed fed was commercial adult broiler feed . Healthy adult chickens were kept in a temperature-controlled room with natural light (20 ± 1°C) , with free access to food and water. The chickens were kept and observed for 7 days. For sample collection, the birds were euthanized by cervical dislocation after intravenous administration of 3% sodium pentobarbital. The left oviduct was extracted immediately. The utero-vaginal junction was taken and fixed in 2.5% glutaraldehyde for transmission electron microscopy method. The utero-vaginal junction was fixed in 4% paraformaldehyde/PBS overnight for Masson staining. The utero-vaginal junction was fixed in the 4% paraformaldehyde/DEPC  above 12 h for fluorescent Tissue sections were dewaxed, stained with Weigert ferroxylin staining solution, and differentiated with acidic ethanol differentiation solution. After sections were washed with water, the Masson blue solution returned to blue, followed by the lichunsin fuchsin staining solution, and then washed with molybdenum phosphomolybdic acid solution and the prepared weak acid working solution. Sections were dyed in aniline blue solution and washed in weak acid working solution. Dehydration was performed in ethanol and xylene  and then cover-slipped . The stained sections were analyzed using a light microscope  equipped with a camera .For FISH, firstly, the gene sequence of chicken CD34 and C-kit was obtained from GenBank, and the GenBank login number was GI: 2024484067(CD34), 303532(C-kit); the DNA STAR software was used to compare it with CD34 and C-kit gene sequences of other species to find the homologous sequence. BLAST analysis was performed on the NCBI website based on chicken the genome database to confirm the specificity of the homologous sequence, and then a specific reverse-phase three-oligonucleotide probe was designed. Fluorescence probe CD34 and C-Kit were synthesized by Wuhan Servicebio Technology Co., Ltd. The tissue sections were dewaxed, washed in DEPC dilution, followed by retrieval of the antigen epitope, and naturally cooled. Add proteinase K (20 μg/ml)  working solution to cover objectives and incubate at 37°C for 30 min. Wash in pure water, then wash three times in PBS (pH 7.4)  on a Rocker device, 5 min each. Add pre-hybridization solution to each section and incubate for 1 h at 37°C. Remove the pre-hybridization solution, add the fluorescence probe CD34 and C-kit hybridization solution with a concentration of 0.25 μg/ml, and incubate the section in a humidity chamber and hybridize overnight at 42°C. Remove the hybridization solution. Wash sections in 2 × SSC  for 10 min at 37°C. Wash sections in 1 × SSC two times for 5 min each at 37°C, and wash in 0.5 × SSC for 10 min at room temperature. Fluorescent staining was protected from light and sections were mounted with a mounting medium with 4′,6-diamidino-2-phenylindole (DAPI) . The sections were examined using a luminescence microscope  equipped with a camera .3) were fixed in 2.5% glutaraldehyde PBS solution pre-cooled overnight at 4°C. Tissue fragments were then rinsed with 0.01 M PBS solution (pH = 7.4) and then post-fixed with 1% osmium tetroxide at room temperature for 1 h . Dehydration was done in ethanol; the material was embedded in Epon812 epoxy resin and polymerized at 60°C for 3 days. After the careful orientation of the 1-μm semi-thin section, the ultrathin sections (50 nm) were prepared and attached to the copper net. The ultrathin sections were stained with 1% uranyl acetate for 10 min and Reynold's lead citrate for 5 min. Ultrathin sections were analyzed with a Hitachi H-7650 transmission electron microscope (Japan).Small sections of fresh tissue  showed that TCs were present in both the muscularis and lamina propria of the utero-vaginal junction and had a typical TCs cell structure –7. The mvia Tps .Since TCs were discovered by Professor Popescu in 2005, they have attracted extensive attention from many researchers. The markers expressed in TCs vary from tissue to tissue, and even from cell to cell within the same tissue . ConnectIn this study, a combination of TEM and FISH (CD34 and C-kit) was used , 26 to rOur TEM results show two or three slender Tps were observed on TEM. The number of Tps depended on the angle and position of the section, as all Tps cannot be completely present on a plane during sectioning , 28. TheThe unique morphology and structure of TCs must allow for unique function. TCs may play a role in signaling between cells. The stroma is composed of cells that integrate all information from the blood vessels, nerves, the immune system, and stem cells through contact with the same or different cells . TCs useTCs were located around capillaries and connected with fibroblasts and pericytes. TCs also express growth factor receptors involved in neovascularization –39. TCs The ICCs in the gastrointestinal tract have the role of slow-wave pacing and conduction . TCs forTCs have also been found in the intestinal crypt of the exocrine pancreas and intestinal stem cells , 50. TCsIn conclusion, our results confirm the presence of TCs at the utero-vaginal junction in chickens. TCs were CD34/C-kit positive. TCs surrounds glands, forming homogenous or heterogenous connections with lymphocytes, plasma cells, smooth muscle cells, blood vessels, collagen fibers, and fibroblasts around them, forming a complex three-dimensional network structure. This study provides morphological basis for the possible role of TCs in regulating utero-vaginal junction physiological function and intercellular communication.This is the first study to confirm the existence of TCs at the utero-vaginal junction of the chicken. In the lamina propria, the TCs were curved and folded, embedded between bundles of collagen fibers and around blood vessels, and released vesicles. In the smooth muscle layer, TCs were mostly distributed around smooth muscle bundles. TCs and their surrounding structures, such as sperm storage tube, smooth muscle cells, blood vessels, collagen fibers, plasma cells, and fibroblasts, formed homotypic and allotypic connections to produce a 3D network structure. We postulate that TCs may play a role in information transmission, which provides a theoretical basis for the relationship between TCs in the utero-vaginal junction and sperm storage, transport, and oviduct disease, and provides a reference for the plasticity of TCs. This study provides morphological basis for the possible role of TCs in regulating the utero-vaginal junction physiological function and intercellular communication.The original contributions presented in the study are included in the article/supplementary material, further inquiries can be directed to the corresponding author/s.The animal study was reviewed and approved by the Science and Technology Agency of Jiangsu Province. Written informed consent was obtained from the owners for the participation of their animals in this study.XZ, QW, PP, and PY: conceptualization, data curation, and writing—review and editing. ZW and BP: formal analysis. ZW, BP, XM, YF, and PY: methodology. XZ, QW, and PP: writing—original draft. All authors have read and agreed to the published version of the manuscript.This work was supported by the National Natural Science Foundation of China (Grant No. 31772688), and the Priority Academic Program Development of Jiangsu Higher Education Institutions, China.The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest.All claims expressed in this article are solely those of the authors and do not necessarily represent those of their affiliated organizations, or those of the publisher, the editors and the reviewers. Any product that may be evaluated in this article, or claim that may be made by its manufacturer, is not guaranteed or endorsed by the publisher.


Although the detrimental effect of red meat on colorectal cancer (CRC) incidence has been extensively reported, no previous studies have comprehensively linked different meat subtypes with colorectal polyp occurrence. The aim was to assess the association of meat and subtypes with colorectal polyp prevalence for the high-risk CRC Chinese population. Besides, we also focused on the association according to sizes, subsites, and multiplicity of polyps.High-risk CRC patients aged 40–80 years were enrolled into the Lanxi Pre-colorectal Cancer Cohort (LP3C) between March 2018 and December 2019. Cross-sectional analyses were conducted by using the baseline data from LP3C. A validated food frequency questionnaire (FFQ) was employed to collect dietary information. Odds ratios (ORs) and 95% confidence intervals (95% CIs) of colorectal polyp prevalence were estimated by multivariate logistic regression.Pfor trend = 0.01) but was not linked to total colorectal polyps after multivariable adjustment. For meat subtypes, higher poultry consumption was significantly related to a higher polyp prevalence . Processed red meat intake was linked to an increased small polyp prevalence (Pfor trend = 0.03) while unprocessed red meat had a relation with a higher rectum polyp prevalence (Pfor trend = 0.04). Furthermore, seafood intake had a significant association with a higher multiple polyp prevalence .2,064 colorectal polyp cases were identified among 6,783 eligible participants in the survey of LP3C (March 2018 and December 2019). Total meat intake was positively related to rectum polyp prevalence (The finding was that poultry meat consumption was related to a higher polyp prevalence. Besides, total meat consumption, processed and unprocessed red meat consumption, seafood consumption had a positive relation with certain polyp subtypes prevalence. Generally recommending reducing total meat consumption, including poultry, processed and unprocessed red meat, and seafood intake, may prevent colorectal polyps. It was announced that one of the top five causes of cancer-related death was colorectal cancer (CRC) in China. Meanwhile, China is undergoing a cancer transition period with an increasing burden of CRC . IncreasColorectal adenomas and polyps that were regarded as precursor lesions of CRC, attracted more and more attention. More and more studies shed light on the risk factors of adenomas and polyps. Higher total meat consumption and higher red meat consumption were both positively related to an increased hyperplastic polyp risk . FurtherAmong all subtypes of meat, the relation of processed red meat or total red meat with polyp risk was widely examined, whereas few previous population-based studies have comprehensively explored the relation of poultry intake or fish consumption with polyp risk. No relation of poultry meat consumption and fish consumption with colorectal adenoma risk was found in a meta-analysis , while aImportantly, previous studies were largely conducted in Western populations whereas little was known among Asian populations, such as Chinese undergoing “Westernization” of diets. Therefore, evidence is needed for Chinese individuals about the role of meat and meat subtypes consumption on colorectal polyp risk. With the aim of filling the gap in the knowledge, we employed data from the Lanxi Pre-colorectal Cancer Cohort (LP3C) to investigate the relation of meat consumption with colorectal polyp prevalence.The LP3C study began in March 2018 in Lanxi, Jinhua, Zhejiang Province, China. High-risk CRC patients aged 40–80 years were enrolled from the Lanxi of 16 government-administered units. Participants were required to complete assessment questionnaire of the CRC risk factor, which included a personal history of colorectal polyps or CRC, family history of CRC, and a fecal occult blood test. Each participant was required to complete two fecal occult blood tests with an interval of a week. The participants who had family or personal history and/or received a positive outcome from the above fecal test were considered as individuals at a high risk of CRC . They wen = 7), aged < 40 or > 80 (n = 40), missing age (n = 3), missing BMI (n = 5) and missing colonoscopy results (n = 210), adenocarcinoma or other malignant tumors patients (n = 19), and implausible energy intake (n = 1). Finally, 6,783 eligible participants were included in the cross-sectional analysis  . The survey enrolled 7,068 participants in total. Exclusion criteria: withdrew (5 women) .A food frequency questionnaire (FFQ) of China Kadoorie Biobank was modified by adding three food groups , and then we used the modified FFQ to achieve dietary data . The 15 2, never smoking, a healthy diet score >40th percentile, physical activity for ≥ 30 min/d of moderate-to-vigorous intensity activity, and alcohol intake . The sum of the five components score was made of a healthy lifestyle. If any one of the above scoring criteria was qualified, participants would be given one point. The range of healthy lifestyle score was 0–5.Detailed information  were also obtained through self-reporting. Anthropometric methods were used to measure weight and height. The Compendium of Physical Activities aided us to calculate metabolic equivalent task hours per week (MET-h/wk) of physical activity . Body weExperienced and qualified proctologists performed electronic colonoscopy. Polyps would be normally removed when proctologists detected colorectal polyps (≥ 5 mm) during the electronic colonoscopy process. But, it would be not extirpated for poor bowel preparation, anticoagulant drug use, and patients with harsh refusal. Pathologic records of pathological sections were acquired by the Pathology Detection Department. The polyps were classified as proximal polyps , distal polyps (descending or sigmoid colon), and rectal polyps (rectum or rectosigmoid junction). The diameter of polyp < 10 mm was deemed as small polyps and diameter ≥ 10 mm was large polyps according to polyp size . Colorec–1⋅d–1) of individual food intake was expressed via the well-known nutrient density method , with cancers, or with extreme energy , respectively. In order to assess whether a divergent association existed or not, subgroup analyses were also performed. The subgroup was classified according to sex , age (< 60 or ≥60 years), BMI (< 24 or ≥24 kg/m2), smoking status (non-smokers or smokers), physical activity (< median or ≥median), alcohol intake (non-drinkers or drinkers), healthy lifestyle score (< median or ≥median) and healthy diet score (< median or ≥median).We further assessed the relation of meat intake with different polyp prevalence according to anatomic location, size, and multiplicity. Sensitivity analyses were performed, including additional adjustment for other underlying confounders  on the basis of model 4. The ORs and 95% CIs of meat consumption were computed after excluding participants with extreme BMI . A positive association was found for poultry meat consumption in the model 1 . The relation was still significant after additional adjustment for demographic factors  and was almost unchanged in model 3 . A significant relation of poultry meat consumption with polyp prevalence was still observed in model 4 . Higher processed red meat consumption was marginally related to a higher colorectal polyp prevalence , whereas a similar association was not detected for total red meat (Pfor trend = 0.81) and unprocessed red meat (Pfor trend = 0.71). Seafood consumption was not related to colorectal polyp prevalence : 1.48 (1.03–2.11); Pfor trend = 0.01]. Furthermore, a marginal associations with rectum polyp prevalence were detected for total red meat consumption  and unprocessed red meat .In anatomic subsites of polyps, it was diagnosed 866 cases of proximal polyps, 900 cases of distal polyps, and 298 cases of rectum polyps . A positQ4vs.Q1 (95% CI): 1.17 (0.99–1.38); Pfor trend = 0.03]. For poultry, participants in the highest consumption also had a higher small polyp prevalence  whereas no relation was found for large polyps  (Q4vs.Q1 (95% CI): 1.70 (1.31–2.21); Pfor trend < 0.001]  and the relation of processed red meat consumption was similar in subgroups (P for interaction >0.05), which indicated baseline characteristics did seem to modify the documented associations. Noticeably, the association of poultry meat intake and polyps prevalence tended to be stronger among the elders aged ≥60 (P for interaction = 0.05)  .In the current analyses, participants with higher poultry meat consumption had a higher colorectal polyp prevalence, especially multiple polyp prevalence and small polyp prevalence. A marginal relation of processed red meat intake with polyp prevalence was found. Unprocessed red meat consumption and total meat consumption were related to a higher prevalence of rectum polyps.Previous studies mainly focused on the role of processed meat intake or red meat intake and few studies detected the relation of various meat subtype consumption with colorectal polyp prevalence. The detrimental role of higher processed meat consumption and higher red meat consumption on colorectal adenoma risk was reported in several meta-analyses, and a dose-response relationship was found with the increase of red meat (100 g/day) or processed meat (50 g/day) , 7, 22. Except for meat-derived mutagens and carcinogens, there were high proteins and important micronutrients  in red meat . DietaryNo consensus has been reached regarding the relationship between fish and poultry consumption (referring to white meat) and colorectal polyp risk. According to the results of Nurses’ Health Study II, fish consumption was not related to colorectal adenoma prevalence [OR 95% CI): 0.96 (0.78–1.17)] whereas adolescence with higher poultry meat consumption had a lower total colorectal adenoma risk [OR (95% CI): 0.80 (0.64–0.99)] . Nonethe5% CI: 0.Q5vs.Q1 (95% CI): 0.45 (0.27–0.75); Pfor trend = 0.005], but not colon adenomas : 2.38 (1.21–4.68); P < 0.05] : 0.66 0.44–0.99)], were related to a lower odds of advanced polyps . A higheadenomas . The abo–0.99], wadenomas , and fisadenomas  intake hadenomas . Besides < 0.05] . In addi < 0.05] .There are several advantages in the current study. The diagnosis of colorectal polyps included detailed histopathologic information, which allowed us to separately evaluate subsites, sizes, and multiplicity of polyps. Moreover, reverse causality was also minimized by assessing dietary factors and other key confounders such as a history of family colorectal cancer before an exam of personal electronic colonoscopy was taken. Most importantly, it was the first time to explore the relation of various subtypes of meat intake with colorectal polyps prevalence among the Chinese population.Several limitations should be noted in the current analysis. First, colorectal adenomas and polyps were not analyzed individually, which would not provide more insight into the different progressing stages of CRC. Nonetheless, we further analyzed the association of subtypes of meat with small and large polyps, which could predict the possibility of a polyp developing into advanced neoplasia. Second, considering the observational study nature, residual or unmeasured confounding cannot be fully discarded. Third, the significant association may be diluted to the null relationship by unavoidable measurement errors. Fourth, although different seafood have different effects for human health, we did not separately collect dietary data of fish, shrimp, and shellfish and could not detect the effect of seafood subtypes intake on the occurrence of polyps. In addition, given that different ethnic groups have distinct dietary patterns and lifestyles, the generalization of our findings to other populations may be limited. Finally, given the observational study nature, a causal relationship cannot be achieved.Our current study investigated the relation of different subtypes of meat intake with the prevalence of polyps among the high-risk CRC Chinese population. The finding was that higher unprocessed red meat intake and total meat intake were related to a higher prevalent rectum polyp prevalence. Higher poultry consumption and processed red meat consumption were both related to an increased small polyp prevalence. Besides, a higher intake of poultry meat had a positive relationship with higher total colorectal polyp prevalence. Seafood consumption had a positive correlation with a higher multiple polyp prevalence. These findings provide emerging evidence supporting the current dietary guideline that recommends the low intake of poultry, processed and unprocessed red meat, and seafood for colorectal polyps prevention among the high-risk CRC Chinese population.The raw data supporting the conclusions of this article will be made available by the authors, without undue reservation.The studies involving human participants were reviewed and approved by the Ethical Committee of Lanxi Red Cross Hospital. The patients/participants provided their written informed consent to participate in this study.XC, ZY, PH, ST, and WZ: data collection. YL and FW: analysis and manuscript writing. YL, FW, XL, and PZ: data interpretation. JJ: study design. YZ, WZ, and JJ: critical revision of manuscript. All authors contributed to the article and approved the submitted version.The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest.All claims expressed in this article are solely those of the authors and do not necessarily represent those of their affiliated organizations, or those of the publisher, the editors and the reviewers. Any product that may be evaluated in this article, or claim that may be made by its manufacturer, is not guaranteed or endorsed by the publisher.


On July 15, 2021, the Secretary of Health of Honduras (SHH) was notified of an unexpected number of mucormycosis cases among COVID-19 patients. SHH partnered with the Honduras Field Epidemiology Training Program, the Executive Secretariat of the Council of Ministers of Health of Central America and the Dominican Republic (SE-COMISCA), Pan American Health Organization (PAHO), and CDC to investigate mucormycosis cases at four geographically distinct hospitals in Honduras.Mucormycosis is a severe, often fatal disease caused by infection with angioinvasive molds belonging to the order Mucorales. Risk factors for mucormycosis include certain underlying medical conditions  and the use of certain immunosuppressive medications ,Given the severe outcomes associated with mucormycosis, clinicians should remain vigilant for this disease during the COVID-19 pandemic, including in immunocompetent patients. Early mucormycosis diagnosis is possible, even in resource-limited settings (


Flavonoids are an important class of secondary metabolites widely found in plants, contributing to plant growth and development and having prominent applications in food and medicine. The biosynthesis of flavonoids has long been the focus of intense research in plant biology. Flavonoids are derived from the phenylpropanoid metabolic pathway, and have a basic structure that comprises a C15 benzene ring structure of C6-C3-C6. Over recent decades, a considerable number of studies have been directed at elucidating the mechanisms involved in flavonoid biosynthesis in plants. In this review, we systematically summarize the flavonoid biosynthetic pathway. We further assemble an exhaustive map of flavonoid biosynthesis in plants comprising eight branches  and four important intermediate metabolites . This review affords a comprehensive overview of the current knowledge regarding flavonoid biosynthesis, and provides the theoretical basis for further elucidating the pathways involved in the biosynthesis of flavonoids, which will aid in better understanding their functions and potential uses. Flavonoids comprise a group of phenylpropanoids that, as water-soluble pigments, are stored in the vacuoles of plant cells . Except Some flavonoids play an important role in plant development and defense. Flavonoids constitute one of the main pigments in plants, such as anthocyanins ; chalcones and aurones (yellow pigments); and flavonols and flavones , which impart on plants a wide variety of colors . FlavonoFlavonoids have long been a major focus of research into secondary metabolism. On PubMed, performing a search using ‘flavonoid’ as a search term retrieves more than 10,000 articles in both 2019 and 2020. Recent decades have witnessed a considerable renewed interest in flavonoid biosynthesis in plants. In this review, we present a systematic summary of what is known of the flavonoid biosynthetic pathway in plants, presenting a model of flavonoid biosynthesis that includes eight branches and four intermediate metabolites , therebyp-coumaroyl-CoA through the activity of phenylalanine ammonia lyase (PAL), cinnamic acid 4-hydroxylase (C4H), and 4-coumarate: CoA ligase (4CL). PAL catalyzes the first committed step in the general phenylpropanoid pathway, namely, the deamination of phenylalanine to trans-cinnamic acid [StlA, a Photorhabdus luminescens PAL-encoding gene, was shown to be involved in the production of a stilbene antibiotic [trans-cinnamic acid to generate p-coumaric acid. This is also the first oxidation reaction in the flavonoid synthesis pathway [Populus trichocarpa and Arabidopsis thaliana, the expression level of C4H has been associated with the content of lignin, an important phenylpropanoid metabolite [p-coumaroyl-CoA by the addition of a co-enzyme A (CoA) unit to p-coumaric acid. In plants, the 4CL gene usually exists as a family the members of which mostly display substrate specificity. Of the four 4CL genes in A. thaliana, At4CL1, At4CL2, and At4CL4 are involved in lignin biosynthesis, while At4CL3 has a role in flavonoid metabolism [PAL, C4H, and 4CL are often coordinately expressed [Flavonoids are generated from phenylalanine through the phenylpropanoid pathway, while phenylalanine is synthesized via the shikimate pathway . The firmic acid . Additiomic acid . PAL actmic acid  while Sttibiotic . The sec pathway . In Poputabolite . In the tabolism . In plantabolism , while Pxpressed . The genp-coumaroyl-CoA into the flavonoid biosynthesis pathway represents the start of the synthesis of specific flavonoids, which begins with chalcone formation [p-coumaroyl-CoA and three molecules of malonyl-COA, derived from acetyl-CoA via the activity of acetyl-CoA carboxylase (ACCase), generate naringenin chalcone  through the action of chalcone synthase (CHS) [Solanum lycopersicum), RNA interference (RNAi)-mediated suppression of CHS leads to a reduction in total flavonoid levels [CHR1 gene from Lotus japonicus in petunia leads to the formation of isoliquiritigenin and a decrease in anthocyanin content [The entry of ormation . One molse (CHS) . CHS, a se (CHS) ,27. In td levels . Chalcond levels . Overexp content . Because content ,32. Diff content . Chalcon content .p-coumaroyl-CoA and malonyl-CoA as substrates and catalyzes the formation of the stilbene backbone, such as resveratrol [2) molecules, while CHS catalyzes the formation of C15 skeletons (C6-C3-C6), with only 3 molecules of CO2 being released [Vitis amurensis calli, the overexpression of Picea jezoensis PjSTS1a, PjSTS2, and PjSTS3 greatly increases the total stilbene content [trans-resveratrol  that has undergone various modifications, such as isomerization, glycosylation, methylation, oligomerization, and prenylation [Trans-resveratrol can be converted to polydatin, pterostilbene, and piceatannol by glycosylation, methylation, and hydroxylation, respectively [trans-3′-(3-methyl-2-butenyl)-resveratrol and trans-arachidin-1/2/3 [cis-stilbene are derived from the oligomerization and isomerization of trans-resveratrol, respectively [Stilbene synthase (STS) also uses veratrol ,35. The veratrol ,37. STS,veratrol . Howeverreleased . In Viti content . Most plnylation . Trans-rin-1/2/3 . Viniferectively ,41.O-glucosyltransferase (CH4′GT) catalyzes the formation of THC 4′-O-glucoside from THC in the plant cytoplasm. The former is then transferred to the vacuole and converted to aureusidin 6-O-glucoside (aurone) by the action of aureusidin synthase (AS) [Antirrhinum majus and Dahlia variabilis [O-glucoside) by CH4′GT and/or AS [Ipomoea nil [Torenia [AmCH4′GT and AmAS1 genes leads to the accumulation of aurone 6-O-glucoside. Furthermore, various classical substitution patterns, such as hydroxylation, methoxylation, and glycosylation, lead to the formation of a series of aurone compounds, with over 100 structures having been reported to date [Aurones, important yellow pigments in plants, comprise a class of flavonoids derived from chalcone . Aurone ase (AS) ,46. AS criabilis . 2′,4′,6nd/or AS ,47. CH4′nd/or AS . AS, a hnd/or AS . Both inmoea nil  and Tore[Torenia , the co- to date .CHI was found to be positively correlated with flavonoid content in A. thaliana [Dracaena cambodiana and tobacco, the overexpression of DcCHI1 or DcCHI4 leads to increased flavonoid accumulation [CHI enhances the level of chalcone in pollen [CHI catalyzes the intramolecular cyclization of chalcones to form flavanones in the cytoplasm, resulting in the formation of the heterocyclic ring C in the flavonoid pathway ,51. In gthaliana . In bothmulation . In trann pollen . Furthern pollen . Flavanon pollen .2+-dependent dioxygenases mainly found in members of the Apiaceae [Morus notabilis FNSI can use both naringenin and eriodictyol as substrates to generate the corresponding flavones [A. thaliana, the overexpression of Pohlia nutans FNSI results in apigenin accumulation [FNSII were reported to be consistent with flavone accumulation patterns in the flower buds of Lonicera japonica [Medicago truncatula, meanwhile, MtFNSII can act on flavanones, generating intermediate 2-hydroxyflavanones (instead of flavones), which are then further converted into flavones [C-glycosyl flavones . Naringenin and eriodictyol are converted to apigenin C-glycosides and luteolin C-glycosides under the action of flavanone-2-hydroxylase (F2H), C-glycosyltransferase (CGT), and dehydratase [Flavone biosynthesis is an important branch of the flavonoid pathway in all higher plants. Flavones are produced from flavanones by flavone synthase (FNS); for instance, naringenin, liquiritigenin, eriodictyol, and pentahydroxyflavanone can be converted to apigenin, dihydroxyflavone, luteolin, and tricetin, respectively ,59,60. FApiaceae . MeanwhiApiaceae ,64. FNS flavones . In A. tmulation . The expjaponica . In Mediflavones . Flavanoydratase .Scutellaria baicalensis is a traditional medicinal plant in China and is rich in flavones such as wogonin and baicalein [S. baicalensis, namely, the general flavone pathway, which is active in aerial parts; and a root-specific flavone pathway [O-methyl transferases (OMTs) [aicalein . There a pathway ), which  pathway . In this pathway . Subsequ pathway . Chrysin pathway . Norwogos (OMTs) . Additios (OMTs) . The aboO-methyl transferase (IOMT) [Glycine max IFS in Allium cepa led to the accumulation of the isoflavone genistein in in vitro tissues [IFS1 gene using CRISPR/Cas9 led to a significant reduction in the levels of isoflavones such as genistein [The isoflavone biosynthesis pathway is mainly distributed in leguminous plants . Isoflave (IOMT) . IFS ande (IOMT) ,80. IFS,e (IOMT) . The ove tissues . Knockinenistein . Variousenistein ,83. Maloenistein ,85.Sinningia cardinalis and Zea mays [Besides flavones and isoflavones, the biosynthesis of phlobaphenes also uses flavanones as substrates . PhlobapZea mays .Dihydroflavonol (or flavanonol) is an important intermediate metabolite and a key branch point in the flavonoid biosynthesis pathway. Dihydroflavonol is generated from flavanone under the catalysis of F3H and is the common precursor for flavonol, anthocyanin, and proanthocyanin ,96. F3H F3H leads to the generation of DHK in tobacco and yeast [Silybum marianum, F3H was shown to catalyze the synthesis of taxifolin (DHQ) from eriodictyol [AgF3H was significantly positively correlated with DHM content in different tissues of Ampelopsis grossedentata [F3H, a FeⅡ/2-oxoglutarate-dependent dioxygenase, catalyzes the dydroxylation of flavonones at position C-3 and is the key enzyme in dihydroflavonol synthesis . Becausend yeast . In Silyodictyol , while tedentata .Glycine soja f3′5′h mutant [F3′H and F3′5′H, both cytochrome P450 enzymes, catalyze the hydroxylation of flavonoids at position C-3′ or C-3′ and C-5′ of ring B, respectively, so as to the formation of substrates of different pathways ,103. F3′h mutant .Camellia sinensis FLSa/b/c in tobacco promoted the accumulation of kaempferol and a decrease in anthocyanin content in flowers [FLS of Allium cepa in tobacco enhanced quercetin signals in the roots [Flavonols are flavonoid metabolites that are hydroxylated at position C-3 of ring C . Their C flowers . Meanwhihe roots .Vitis vinifera, DFR converts DHK to leucopelargonidin [Brassica oleracea DRF1 was shown to promote anthocyanin accumulation, whereas the virus-induced silencing of the BoDRF1 gene elicited the opposite effect [DFR and FLS inhibit each other’s transcription [DFR, a NADPH-dependent reductase, is the key enzyme in flavonoid metabolism in the anthocyanidin and proanthocyanidin pathway and catalyzes the formation of a hydroxyl group at position C-4 of ring C ,115,116.rgonidin . The ovee effect . DFR andcription .ANS overexpression in strawberry enhanced the anthocyanin concentration [Leucoanthocyanidin is an important intermediate product in the flavonoid pathway and the direct synthetic precursor of anthocyanidin and proanthocyanidin. The colorless leucopelargonidin, leucocyanidin, and leucodelphinidin are transformed into the corresponding anthocyanidins  under the catalysis of anthocyanidin synthase (ANS), also known as leucoanthocyanidin dioxygenase (LDOX) ,123. Likntration . In planntration ,127. OMTntration ,128. Thentration ,118,127.In addition to the above-mentioned anthocyanins, a rare type of anthocyanin, 3-deoxyanthocyanidin, also exists in plants . The biotrans-flavan-3-ols, afzelechin, catechin, and gallocatechin, respectively [cis-flavan-3-ols, epiafzelechin, epicatechin, and epigallocatechin [trans-flavan-3-ols, and cis-flavan-3-ols are the basic proanthocyanidin units. Proanthocyanidins are synthesized via the polymerization (or condensation) of flavan-3-ols [Populus tomentosa, the overexpression of LAR3 greatly increased the proanthocyanidin levels [OvBAN, an ANR gene from Onobrychis viviaefolia, in alfalfa (Medicago sativa) promoted ANR enzyme activity and enhanced proanthocyanidin content [Proanthocyanidins, also known as condensed tannins, are an important type of flavonoid synthesized from leucoanthocyanidins and anthocyanidins. Leucoanthocyanidin reductase (LAR) converts leucoanthocyanidins, leucopelargonidin, leucocyanidin, and leucodelphinidin to ectively ,136. LARectively . Anthocycatechin . ANR is catechin . Flavan-an-3-ols ,141. Colan-3-ols  and can an-3-ols . LAR andn levels . The ect content . Because content .AN4 (a R2R3-MYB-encoding gene) can enhance anthocyanin biosynthesis by promoting the expression of anthocyanin biosynthesis genes, such as CHS, CHI, F3H, and DFR [Cucumis sativus, the R2R3-MYB transcription factor CsMYB60 induced the expression of CsFLS and CsLAR by binding to their promoters, thereby promoting flavonol and proanthocyanidin biosynthesis [Malus domestica), MdMYB15L was reported to interact with MdbHLH33 and inhibit the promotion of the MdbHLH33-MYB-WD40 (MBW) complex, thereby also suppressing anthocyanin biosynthesis [Transcriptional control plays a central role in the modulation of flavonoid biosynthesis . The MBW and DFR . In Cucuynthesis . MYB traynthesis .DhbHLH1 induces anthocyanin synthesis in the white petals of Dendrobium hybrids [Dianthus caryophyllus, meanwhile, the “red speckles and stripes on white petals” phenotype results from the local expression of bHLH, which promotes the expression of DFR and that of downstream enzymes in the anthocyanin biosynthetic pathway [bHLH transcription factors have been shown to participate in the regulation of flavonoid biosynthesis. The transient expression of  hybrids . In Dian pathway .FLS expression [WD40, widely present in eukaryotic cells, contains multiple tandem repeats of a WD motif and interacts with other proteins through its WD domain . Generalpression .CHS1 gene, thereby regulating flavonoid synthesis and affecting tomato peel color [MYB12, FLS, and UGT and activating their expression; under shading, meanwhile, PIF3 inhibited flavonol accumulation by activating the expression of MYB7, which encodes a transcriptional repressor [MYB10.1, while NAC1 was repressed by SPL1 [SlDFR promoter and directly activate its expression, which augments anthocyanin biosynthesis, while SlCSN5, a subunit of the COP9 signalosome, induces the degradation of SlBBX20 by enhancing its ubiquitination [MdLOB52 and further activating its expression [Also in tomato, besides the MBW complex, the transcription factors NF-YA, NF-YB, and NF-YC can reportedly form a NF-Y protein complex that binds to the promoter of the el color . Additioel color . In the epressor . In peac by SPL1 . In the  by SPL1 . The BTB by SPL1 . SlBBX20tination . MdARF19pression . BES1, apression F3′5′H from Campanula medium (Canterbury bells) and A3′5′GT  from Clitoria ternatea (butterfly pea) driven by the native (Chrysanthemum morifolium) F3H promoter induced the synthesis of delphinidin and generated true blue Chrysanthemums [F3H, FLS, and OMT in Corynebacterium glutamicum yielded pterostilbene, kaempferol, and quercetin at high concentrations and purity [Escherichia coli, cyanidin 3-O-glucoside was generated through the induction of ANS and 3GT using a bicistronic expression cassette [Flavonoids are abundantly present in land plants where they have diverse functions; as dietary components, they also exert a variety of beneficial effects in humans ,164,165.nthemums . Flavonod purity . In Eschcassette . These oOver the past few decades, flavonoid biosynthesis has been among the most intensively investigated secondary metabolic pathways in plant biology, and a considerable number of studies have contributed to revealing the exquisite mechanisms underlying the biosynthesis of flavonoids in plants ,135. HowS. baicalensis, which generated root-specific flavones such as baicalein and norwogonin [Plants are rich in diversity and often produce specific secondary metabolites. Recent studies have identified a unique flavone synthesis pathway in the root of the medicinal plant rwogonin ,70,71. Aflavonoid 7-O-glycosyltransferase (OsUGT706C2) gene with a role in modulating flavonol (kaempferol) and flavone (luteolin and chrysoeriol) metabolism was identified by metabolite-based genome-wide association analysis [Camellia oleifera [In addition, combined multi-omics  analysis provides a direction for the study of plant synthetic biology. In rice, a analysis . Proteomoleifera . The con


In the hours following noise exposure, there was a significant increase in cochlear mRNA expression of NOX3 in wild type animals. By using RNAscope in situ hybridization, NOX3 expression was primarily found in the Rosenthal canal area, colocalizing with auditory neurons. One day after the noise trauma, we observed a high frequency hearing loss in both knock-out mice, as well as their wild type littermates. At day seven after noise trauma however, NOX3 and p22phox knockout mice showed a significantly improved hearing recovery and a marked preservation of neurosensory cochlear structures compared to their wild type littermates. Based on these findings, an active role of NOX3 in the pathophysiology of noise-induced hearing loss can be demonstrated, in line with recent evidence obtained in other forms of acquired hearing loss. The present data demonstrates that the absence of functional NOX3 enhances the hearing recovery phase following noise trauma. This opens an interesting clinical window for pharmacological or molecular intervention aiming at post prevention of noise-induced hearing loss.The reactive oxygen species (ROS)-generating NADPH oxidase NOX3 isoform is highly and specifically expressed in the inner ear. NOX3 is needed for normal vestibular development but NOX-derived ROS have also been implicated in the pathophysiology of sensorineural hearing loss. The role of NOX-derived ROS in noise-induced hearing loss, however, remains unclear and was addressed with the present study. Two different mouse strains, deficient in NOX3 or its critical subunit p22 Hearing loss is the most common neurosensory deficit in humans and the third most prevalent chronic disability over 65 years old, affecting over 460 million people world-wide . Noise eOver the past decades it became clear that oxidative stress is a common denominator of many forms of acquired sensorineural hearing loss . Noise-iphox, have a similar vestibular phenotype, comparable to the head-tilt phenotype of NOX3 mutant mice (Growing evidence suggests that the reactive oxygen species (ROS) generating NADPH oxidase enzyme NOX3 is an important mediator of oxidative damages and a possible common mediator in several forms of sensorineural hearing loss . NADPH oant mice . Althougant mice  and NOX3ant mice . Howeverant mice . In thesant mice . Noise iant mice . Counterant mice . TherefoNox3het−4J/J) (phox knockout (C57BL/N Cyba knockout)   and the nockout) . The p22activity . Our resactivity  or molecactivity  intervenin situ hybridization  where not significantly affected by the noise stimulus , both deficient in NOX3 activity. Our results demonstrate a partial, but statistically significant protection against noise-induced hearing loss along with preserved cochlear cellular structures in both mouse strains. These findings are in line with previous studies on two other forms of acquired hearing loss, namely the drug-induced and age-associated hearing loss  in the pathophysiology of noise induced hearing loss. Furthermore, Nox1 was not detectable in cochlear samples by real-time qPCR and neither Nox2 nor Nox4 were found at significant level in cochlear structures important for hearing, such as auditory neurons, organ of Corti, or stria vascularis, at least by using the RNAscope method  could be offered NOX3 inhibiting treatment in the hours following the insult and expect a better recovery. Whether the commonly used glucocorticoids for these situations today work through inhibition of NOX3, or whether a more specific NOX3 inhibition through pharmacological or molecular intervention is more effective remains to be investigated in the future. In absence of a bona fide NOX3 inhibitor, a nucleotide-based approach seems to be more promising , at leasIn conclusion, our data demonstrates that NOX3 contributes significantly to noise-induced cochlear damage as previously demonstrated for two other forms of acquired forms of sensorineural hearing loss, namely age-related and cisplatin-induced hearing loss. NOX3 activity therefore seems to be a common molecular trigger influencing redox regulated neuronal activity, pathologically altered in different forms of acquired sensorineural hearing loss . This coNox3 mutant mice  were purchased from Jackson laboratory, while p22phox knockout (C57BL/N Cyba knockout) were generated in Prof. Ulla Knaus laboratory  ketamine (10%) and xylazine (5%) anesthesia (dose 10 μL/g). If necessary 10% ketamine solution was injected intramuscularly (dose 5 μL/g) to elongate the anesthesia. For the noise trauma, 6 weeks old mice were subjected to an 8–16 kHz noise band for 2 h at 116–120 dB SPL  before noise exposure (pre-ABR), 24 h (D1) and 7 days (D7) after noise trauma. ABR recordings were performed conform previously described protocols . BrieflyFollowing D7 ABR, anaesthetized mice were sacrificed. Temporal bones were isolated from the mice skull, in order to further dissect the auditory bulla. Dissected cochleae were placed in 4% paraformaldehyde overnight at room temperature. Cochleae were decalcified using USEDECALC solution  under sonication for 48 h . After decalcification, both cochlea from the same animal were arranged for different protocols. While one cochlea was embedded in paraffin for cochlear morphology assessment (Hematoxilin-Eosin staining or RNAscope), the contralateral cochlea was micro-dissected to evaluate the sensory epithelium (cytocochleograms) as previously described .The decalcified cochleae were dissected with microsurgical forceps under a binocular microscope as previously described . BrieflyFollowing standardized protocols, decalcified cochleae were sequentially dehydrated and embedded in paraffin. Mid-modiolar cuts of 5 µm were processed and loaded onto gelatin-coated slides. Adjacent mid-modiolar cuts were employed on hematoxylin/eosin and RNAscope protocols (see sections below).2O and briefly submerged in an alcohol and HCl bath, and finally washed with alcohol 70%. Following Hematoxylin staining, slides were briefly (2–3 s) exposed to the eosin reagent. After staining the slides were dehydrated with alcohol and xylol in successive bath steps, and mounted with the commercial Eukitt mounting medium. Images were obtained with a Zeiss Axioskop 2 plus microscope. Spiral ganglia density was reported and assessed on the resulting images using ImageJ software.Harris’ hematoxylin/eosin staining was performed on 5 non-consecutive slides of each cochlea including all cochlear turns. Mid-modiolar paraffin slides were re-hydrated with successive xylene and alcohol baths, then exposed to Hematoxylin for 5 min, rinsed with H2) within all cochlear turns . Five non-consecutive sections were evaluated for each turn and the densities were averaged. Similarly, cytocochleogram images were employed to determine the number of synapsis/inner hair cell and outer hair cell viability. For synaptic ribbons quantification, images were recorded with a 63X confocal objective, generating 10-15 um Z stack images (0.7 um steps). The resulting file was projected into a single plane to ensure accurate quantification of the ribbons distributed along the Z axis. For each cochlear turn, two different segments of 10–15 inner hair cells were assessed to obtain an average number of ribbon synapses. In the same way, the hair cell viability (cytocochleogram) was evaluated in two representative areas of 100 µm and the survival rate was averaged for each cochlear section . Both Inner hair cells (IHC) and outer hair cells (OHC) were recorded with a 20X confocal objective. The open source ImageJ program was used for the image analysis.Hematoxylin-eosin mid-modiolar cuts images were analyzed on the open source software ImageJ to determine the spiral ganglion neurons density. SGN nuclear quantification was performed and normalized to the Rosenthal’s canal area . The highest normalized relative quantity was designated as a value of 1.0. Fold changes were calculated from the quotient of means of these RNA normalized quantities and reported as ±SEM. Sequences of the primers used are provided in Following RNA purification and tritation, 500 ng of RNA were prepared for cDNA synthesis using the Takara PrimeScript RT reagent Kit, following manufacturer’s instruction [see also ]. Real-tβ-III tubulin  immunostaining were used for Fluorescent multiplex assay. Finally, the slides were dehydrated, cleared and mounted with Tissue-Tek® Glas™ . Samples were visualized with a confocal laser-scanning microscope (Zeiss LSM700) equipped with a CCD camera (Leica Microsystems) employing the Plan-Neofluar 20X/0.50 and Plan-Apochromat 63X/1.4 (Oil) objectives. Pictures were analyzed using the open source software ImageJ.Mid-modiolar cuts (5 µm) obtained from decalcified cochleae (see section above - mid modiolar preparations) were loaded onto gelatin-coated slides and followed RNAscope probe hybridization. RNAscope 2.5 HD Assay–BROWN assay  or Fluorescent multiplex assay  was performed according to manufacturer’s protocol. Selected paraffin sections were hybridized with the probes Mm-NOX3-C1 , Mm-Cybb , Mm-NOX4 , Mm-Ppib-C1  as positive control and DapB-C1  as negative control at 40°C for 2 h and revealed with TSA Opal570 . For BROWN assay, Mayer hematoxyline staining (30 s) was employed as counterstaining agent, whereas DAPI and RNAscope samples were analyzed and quantified employing the open-source software FIJI. RNAscope signal quantification was automatized employing FIJI’s macro functionality together with Ilastik software as segmentation tool. Multi z-stack images were recorded employing a Axiocam Fluo microscope with a 40X EC Plan-Apochromat objective. Multiple tiles comprising the region of interest were acquired and merged employing Zeiss commercial software to generate a single image.Employing FIJI macro software, the resulting z-stack images were projected into a single plane and divided into 2 channels. Blue channel  was processed accordingly to reduce background noise. The red channel  followed a Richardson-Lucy deconvolution  followed by proper processing (background subtraction). Appropriated PSF was provided during each image deconvolution, employing FIJI’s plugin “PSF generator” (macro automatized). The deconvoluted red channel was then segmented following a previously trained Ilastik model, and transformed to a binary image with Huang’s approach. Proper morphometric operators were applied and the RNAscope signal was quantified following FIJI’s “Analyze particle” command (1.5-Infinity pixel).The designed macro then normalized the number of dots quantified on the previous section  to the area of interest . In order to proceed with the normalization, we manually determine the area of interest  following each image blue channel. The results were represented as RNAscope dots/mm2 (except for the Organ of Corti). RGB images were automatically generated during the macro processing, allowing the user to verify the absence of anomalies during the process.p < 0.05 was considered as statistically significant. *p < 0.05, **p < 0.01, ***p < 0.005, ****p < 0.0005.Real time qPCR data were analyzed using One-way ANOVA followed by Dunnett’s multiple comparison test. Other datasets were analyzed using Two-way ANOVA followed by Bonferroni multiple comparison test. GraphPad Prism software (version 8.4.3) was used. Values with All procedures were approved by the local veterinary office and the Commission for Animal experimentation of the Canton of Geneva, Switzerland, authorization number GE-28-18.