As the proportion of older adults in the world’s total population continues to grow, the adverse health outcomes of age-related hearing loss are becoming increasingly recognized. While research has shown that age-related hearing loss is the single greatest modifiable risk factor for dementia, use of hearing aids remains low worldwide, even in many middle- and high-income countries. Reasons for poor uptake of hearing aids are likely to involve a combination of factors, ranging from increasing costs of hearing aid technology to a widespread lack of insurance coverage. This article aims to identify the current state of access to hearing aids, focusing on eight middle- and high-income countries. We discuss how to facilitate greater access to hearing aids for patients by addressing changes in how devices are regulated, technological advancements in hearing devices, the need to adjust reimbursement schemes and the importance of adaptation among the community workforce for hearing-care. Since the 1990s, worldwide efforts to screen for hearing loss in early childhood, followed by appropriate paediatric hearing interventions, have followed from evidence that hearing loss markedly impacts a child’s intellectual, social, and emotional development.,,,This focus on a life-course approach to hearing loss is timely given the projected global increase in the number and relative proportion of older adults over the next 30 years.–,–,Research over the past decade has now demonstrated the broad impact that age-related hearing loss has on the functioning of older adults and future health outcomes.In this article we explore the financing and delivery systems for hearing aids for adults in several countries that are currently facing the challenges of an ageing society. We discuss areas where innovative policies and strategies could be employed to increase access to hearing aids.We gathered data for this article through a wide search of the grey literature from August 2018 to April 2019, which included white papers, government reports, government websites, industry reports and regulatory association guidelines. The search was limited to English language sources. We used personal correspondence with academic and industry experts to address gaps in publicly available data.We focus on eight middle- and high-income countries . We chose these countries to demonstrate variations across five factors that affect how hearing aids and related services are delivered: (i) availability of insurance coverage; (ii) training and scope of practice of hearing-care providers; (iii) pathways for obtaining hearing aids; (iv) where patients obtain their devices; and (v) how sales of hearing aids are regulated .The costs associated with purchase of a hearing aid may be covered by public insurance, private insurance or out-of-pocket payments by the patient at the point of purchase (or combinations of these).,,,,,–Public insurance for hearing aid purchases and services varies widely across countries by the population covered, device options and generosity of benefits. In the United Kingdom, all patients have access to public coverage of the costs of hearing aids whereas Australia and the United States restrict social insurance coverage of hearing aids to older adults who meet qualifying criteria, such as low income.,–Private insurance and out-of-pocket payments are the main means of financial access to hearing aids for a high proportion of patients worldwide .,,,,–,,–The two major categories of hearing aid providers, according to the scope of care required, are audiologists and hearing aid dispensers , with contributions from various other professionals .,,,,,The purchasing pathway refers to the steps through which a patient requiring a hearing aid must proceed, starting from the identification of a deficit in hearing to the point they acquire a hearing aid. In most countries providing public insurance, patients must receive a diagnostic hearing test and consult an audiologist, a general physician or an ear, nose and throat specialist before being eligible to purchase a hearing aid .Other countries are less stringent on the requirement of visiting a hearing-care professional before purchasing hearing aids. In the Netherlands, patients can proceed directly to a hearing-aid dispenser for a hearing assessment and purchase of devices . In Australia, Germany and the United Kingdom, patients who are not receiving public insurance coverage can directly purchase hearing aids from hearing-aid dispensers . China also allows patients who visit hearing-aid shops or clinics outside of hospitals to purchase their devices directly . In Japan, where insurance is non-existent, patients are free to purchase hearing aids at their own discretion .,,One pathway to purchasing devices is at the hospital or office of the hearing-care provider where patients undergo their medical consultations and diagnostic testing. However, patients are increasingly using conveniently located and diversified retail clinics, which range from small private vendors to large chain stores .,In the eight countries examined, hearing aids are classified as medical devices within regulatory bodies that dictate strict criteria that must be met before sale in the market. Current regulations around the sale of hearing aids can limit access for some patients due to the requirement for medical clearance by a designated health professional before purchase. However, it is important to note that while hearing aids are regulated, devices classified as personal sound amplification products are generally not .28,34Box 1Hearing aids are regulated by the United States Food and Drug Administration (FDA) as class I or class II medical devices. They are defined as “any wearable instrument or device designed for, offered for the purpose of, or represented as aiding persons with or compensating for, impaired hearing” (21 CFR 801.420).Personal sound amplification products are over-the-counter electronic devices that are not regulated by the FDA. The FDA has issued guidance that these devices are “intended to amplify environmental sound for non-hearing impaired users… They are not intended to compensate for hearing impairment or to address listening situations that are typically associated with and indicative of hearing loss.”While funding and delivery of hearing care vary substantially from country to country, there are several common areas where innovations could potentially lead to easier access and increased uptake of hearing aids for adults with hearing loss.Current regulations in most countries stipulate that hearing aids can only be sold or dispensed through a licensed provider to maximize the safety and efficacy of devices. For example, with older analogue hearing aids, calibrating and properly fitting a hearing aid could involve crafting a custom ear-mould to minimize audio feedback and manually adjusting trim potentiometers on the hearing aid to set appropriate levels of gain.,,Over the past 10–15 years, however, innovations have been made in digital circuitry and technologies that allow hearing aids to be self-fitted and set to appropriate levels of audio output to avoid hearing damage. Research demonstrates that such hearing aids are both safe and effective for adults with mild-to-moderate hearing loss, but regulatory models in most countries preclude such devices from being sold directly to patients.,,Recent initiatives in the United States from the National Academies of Science, Engineering and Medicine and the White House President’s Council of Advisors on Science and Technology, suggest a new regulatory model to allow for greater access to hearing aids.,A crucial building block for the feasibility of over-the-counter sales regulations in any country is reliable technology. Advances in digital amplification technologies over the past few decades have led to the emergence of products that could meet the needs of a direct-to-consumer amplification device. In the United States, this resulted in a class of unregulated amplification devices called personal sound amplification products, some of which have similar traits as traditional hearing aids .28,43 ThMany of these devices include now-standard digital hearing aid technologies such as noise-reduction algorithms, directionality and linked remote microphones to tackle hearing in difficult situations. Some key innovations too are empowering individuals with hearing loss to benefit better from amplification products. Feedback suppression and advances in durable plastics allow for non-custom ear tips. Additionally, integration with smartphone applications allows for self-adjustment of hearing aids by the user, with the phone delivering a hearing test and the device performing in situ customization to prescriptive targets. Moreover, changes in battery technology allow for rechargeable units that are easier to manipulate.,,,,While previous research revealed that low-cost, inappropriate technology produces poor electroacoustic results, recent studies have demonstrated that some direct-to-consumer devices are technologically similar to traditional hearing aids.For many health systems, the availability of over-the-counter or direct-to-consumer hearing aids has the potential to disrupt the delivery model for hearing aids and hearing services. Systems that pay for hearing-care services independently of devices may be able to adapt more readily than systems (such as in the United States) that operate on a predominantly bundled reimbursement model where the reimbursement of hearing-care services is routinely coupled with the cost of the device. As these systems evolve with the changing distribution landscape, two important factors require consideration.First, ensuring that patients can continue to access hearing-care services independent of the hearing technology used is of importance. Hearing-care services, provided by audiologists or other hearing-care providers, are a fundamental component to successful hearing aid use. These tasks include orienting individuals to the device, providing fitting and customization specific to the patient’s hearing needs, and providing supportive communication strategies and techniques.A second important factor is the reimbursement of new hearing-aid devices in an expanded marketplace. Ensuring that the availability of over-the-counter hearing aids does not lead to the elimination of insurance coverage is important. Given that the most common pathway to purchase hearing aids is through hearing-care providers, policy-makers must consider how to adapt to a wider array of products available for over-the-counter purchase. The extent of change required depends greatly on how the system of reimbursement currently operates. For example, in the United Kingdom, where publicly-funded insurance coverage is widespread and choice of devices covered by insurance is limited, adapting to an expanded hearing aid marketplace will require substantial change.,,In addition to the potential impact of a shifting hearing aid market on the ability for patients to access devices, the persistently low uptake of hearing aids worldwide indicates that the health behaviour of patients plays a major role in hearing care.,,Community-delivered hearing care broadly encompasses care delivered beyond traditional clinic-based services and incorporates task-sharing through paraprofessionals, such as community health workers, health aides or peer mentors. The community health worker model has been widely adapted for use in diverse settings in low- and middle-income countries.Oyendo Bien consists of group aural rehabilitation sessions for older adults with hearing loss who live along the United States–Mexico border, led by promotoras .,Growing efforts around the world are using community-delivered hearing care to older adults. In the United States, two programmes in low-resource settings have been developed. A country’s hearing health-care system plays an important role in how well society can address the global health priority of hearing loss. A steadily ageing global population and the generally low prevalence of hearing aid use makes it imperative that innovations in policy and practice are implemented to meet the evolving challenges that hearing loss will present in the near future. The wide spectrum of delivery models, hearing-care providers and reimbursement models that we have highlighted in this article provides an overview of the current state of hearing health care across higher-income countries and the possibilities for advancement. Innovations in hearing aid regulation, technology, reimbursement schemes and workforce models provide tangible actions for WHO Member States to consider in addressing the growing global burden of hearing loss and making hearing health a political priority.


In order to clarify the central reorganization in acute period of hearing loss, this study explored the aberrant dynamics of electroencephalogram (EEG) microstates and the correlations with the features of idiopathic sudden sensorineural hearing loss (ISSNHL) and tinnitus. We used high-density EEG with 128 channels to investigate alterations in microstate parameters between 25 ISSNHL patients with tinnitus and 27 healthy subjects. This study also explored the associations between microstate characteristics and tinnitus features. Microstates were clustered into four categories. There was a reduced presence of microstate A in amplitude, coverage, lifespan, frequency and an increased presence of microstate B in frequency in ISSNHL patients with tinnitus. According to the syntax analysis, a reduced transition from microstate C to microstate A and an increased transition from microstate C to microstate B were found in ISSNHL subjects. In addition, the significant negative correlations were found between Tinnitus Handicap Inventory (THI) scores and frequency of microstate A as well as between THI scores and the probability of transition from microstate D to microstate A. While THI was positively correlated with the transition probability from microstate D to microstate B. To sum up, the significant differences in the characteristics of resting-state EEG microstates were found between ISSNHL subjects with tinnitus and healthy controls. This study suggests that the alterations of central neural networks occur in acute stage of hearing loss and tinnitus. And EEG microstate may be considered as a useful tool to study the whole brain network in ISSNHL patients. Sudden sensorineural hearing loss (SSNHL) is the sudden-onset and rapidly developed hearing impairment with reduction in hearing of at least 30 dB in no less than three consecutive audiometric frequencies within 72 h . The incIdiopathic sudden sensorineural hearing loss is often accompanied by tinnitus, vertigo, ear fullness, and so on . Among tTo cure ISSNHL subjects with tinnitus effectively, it is necessary to explore the intrinsic mechanism of ISSNHL completely. Previous researches have showed that ISSNHL is correlated with the deviant activity of some central neural networks . The devElectroencephalography (EEG) is a high temporal resolution technique for exploring electrical activity of central brain areas. It records the electrical potentials within the brain through the electrodes on the scalp . So far,At present, several studies have suggested that some central neuropsychiatric diseases have significant changes in EEG microstates, including schizophrenia, dementia, depression, and panic disorder. According to the researches about these neuropsychiatric diseases, EEG microstate analysis may be an effective way to detect the objective indicators, recognize the disease’s severity, design the treatment plan and evaluate the treatment effect . AdditioSo far, knowledge of microstate abnormalities in ISSNHL is limited. To investigate the neurophysiological mechanism of ISSNHL, this study explores the changes in EEG microstates between ISSNHL subjects with tinnitus and healthy subjects. Additionally, it can confirm whether EEG microstate is a useful tool to investigate ISSNHL. With the alterations of nervous activities after ISSNHL and tinnitus, the hypothesis is that the significant alterations of microstates would be found in ISSNHL patients with tinnitus at rest. The significant result can be considered as a vital electrophysiological indicator of ISSNH.The ISSNHL subjects with tinnitus were recruited from the Ear, Nose and Throat Clinic, Sun Yat-sen Memorial Hospital, Sun Yat-sen University, Guangzhou, China. Detailed selection criteria for inclusion and exclusion in the study were:(1)Patients with at least 30 dB idiopathic sensorineural hearing impairment in no less than three consecutive audiometric frequencies within 72 h were recruited. In addition, ISSNHL was diagnosed by pure tone audiometry (PTA) within 72 h of conscious hearing loss or more than 72 h of conscious hearing impairment without appreciable progression of hearing damage.(2)Patients had sought clinical help because of their hearing loss and tinnitus.(3)Patients with acoustic neurinoma, autoimmune hearing loss, Meniere’s disease, depression, or insomnia were excluded.(4)All patients with head trauma, central nervous system disorders, middle ear surgery or pulsatile tinnitus were also excluded.SD = 13.15 years) and 27 healthy controls . A written consent form was signed by all participants after they had been properly informed about the background and purpose of the research. Our research was approved by the Institution Review Board of the Sun Yat-sen Memorial Hospital at Sun Yat-sen University of China.There were 25 ISSNHL patients with tinnitus  was used to evaluate tinnitus severity. THI assessed the perceived level of handicap in tinnitus patients, according to a 0–100 increasing handicap scale . ParticiElectroencephalogram signals were recorded by a high-density EEG with 128 channels and were saved with Electrical Geodesics, Inc. , and a NetAmps 200 amplifier. After understanding the purpose and requirements of the test, subjects were instructed to sit in a comfortable chair and remain calm. Then, participants were required to open the eyes and fixate a cross mark in front of them. A resting EEG for about 7 min was recorded. The sampling rate was set to 1000 Hz. The impedances of all electrodes were kept less than 50 kΩ and the CZ electrode was used as reference.The raw EEG data was preprocessed by EEGLAB 13.0.0 in the toolbox of Matlab. Firstly, the data was re-referenced against the mean reference by all electrodes. The sampling rate of the data was reduced to 500 Hz/s. A notch filter was implemented at 50 Hz and the signals were band-pass-filtered from 0.5 to 80 Hz. Gross artifacts were manually removed by visual inspection. Some artifacts originated from one or a few distinct sources or a limited volume of space were removed. In addition, some artifacts characterized by a particular temporal pattern such as exponential decay were also removed. For example, in the pretreatment of resting EEG (eye-opening state), the largest artifact comes from eye electricity. Our system has a special electrode (E127 and E126) for recording eye electricity, but the effect of eye electricity is not limited to these two electrodes, it has a great influence on the electrode of frontal region. Therefore, we use ICA algorithm to correct it. The advantages of the ICA algorithm can be viewed in the literature , the artMicrostate analysis was done following the procedure described in the study by i(t) is the potential of the i-th electrode at a certain time point. Vmean(t) is the average value of the instantaneous potential across electrodes and the formula is Vmean(t) = Where, K is the number of electrodes in the EEG data. Vive maps . Then, tive maps . After oive maps . The ampt-Test was performed to explore the significant differences of the microstate parameters between two groups. We applied Pearson correlation analysis to evaluate the correlation between microstate features and the characteristics of ISSNHL and tinnitus. A p level of less than 0.05 (two-sided) was considered to be statistically significant.SPSS 19.0 software was used for all statistical analyses. t = −1.263, df = 50, p = 0.212) and gender . t-tests for microstate parameters between the ISSNHL (N = 25) and control (N = 27) groups are given in t-test. Compared with the control group, microstates A had significant decrease in coverage , lifespan , amplitude , and frequency . And microstate B had an increased presence  in frequency for the ISSNHL group compared with the control group. There was no significant difference in microstates C or D (p > 0.05).Independent sample t = 2.978, df = 50, p = 0.004) in the ISSNHL patients compared with healthy subjects. Additionally, the probability of transition from microstate C to microstate B  was increased significantly. No other significant difference in the probability of transition among microstates was revealed.As shown in r = −0.417, p = 0.038) as well as between THI scores and the probability of transition from microstate D to microstate A . While THI was significant positively associated with the transition probability from microstate D to microstate B . No significant correlation was revealed in the alterations of microstate characteristics with hearing threshold in ISSNHL subjects (p > 0.05).The associations between alterations in microstate parameters and tinnitus characteristics were shown in As far as we know, it is the first research to explore the aberrant dynamics of EEG microstate in ISSNHL patients with tinnitus. The purpose of this research was evaluating the early stages of cortical plasticity after ISSNHL and tinnitus by using resting-state EEG microstate. There were significant differences in the temporal characteristics of the EEG microstate between ISSNHL patients and controls.The coverage, lifespan, amplitude, and frequency of microstate A were significantly reduced in our study. Increase in coverage of microstate B was also revealed in the SSNHL subjects with tinnitus. A study by In current study, no significant difference was found in microstate C or microstate D. According to the syntax analysis, a significant reduction in the probability of transition from microstate C to microstate A was found. The reduced transition from the salience network to the auditory network was likely due to the deficiency of auditory sensory input. In addition, the increase was significantly found in the transition probability from microstate C to microstate B. This result indicated the compensatory plasticity after hearing loss with increased activity from the salience network to the visual network. ISSNHL subjects began to rely more on the visual information to compensate for their hearing impairment .Another interesting finding was the negative correlations between THI scores and the frequency of microstate A as well as between THI scores and the transition probability from microstate D to microstate A. While a positive association was found between THI scores and the transition from microstate D to microstate B. These results indicated that the severity of tinnitus was related to the central plasticity after ISSNHL with alterations of auditory network and changes of cognitive function . The decAll in all, the results of this study showed that there was a reduced activity in auditory network and an increased activity in visual network. In addition, the negative correlation between THI scores and frequency of microstate A was found. The findings of this study provided the theoretical basis for the treatment of ISSNHL patients with tinnitus. For example, repetitive transcranial magnetic stimulation (rTMS) is a non-invasive way that can modulate the excitability of the brain cortex . And rTMNonetheless, it is necessary to realize that ISSNHL is a heterogeneous disease with different hearing function, laterality and concomitant symptoms, such as tinnitus and vertigo . TherefoThere were several significant differences in temporal characteristics and syntax of EEG microstates between ISSNHL patients with tinnitus and the controls at rest. This study suggests that the alterations of central neural networks occur in acute stage of hearing loss and tinnitus. EEG microstate may be considered as a useful way to explore the brain network in ISSNHL patients with tinnitus.The datasets generated for this study are available on request to the corresponding author.This study was carried out in accordance with the recommendations of the Institution Review Board of the Sun Yat-sen Memorial Hospital with written informed consent from all subjects. All subjects gave written informed consent in accordance with the Declaration of Helsinki. The protocol was approved by the Institution Review Board of the Sun Yat-sen Memorial Hospital.YxC and SC conceived and designed the research. YhC, JoL, and C-DW performed the experiments. JeL, NH, C-PD, and ML responsible for the data analysis. FZ, YxC, and YZ drafted the manuscript. All authors had a lot of contributions at all stages of preparing the manuscript.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.


Recent studies have shown that the representation of an item in visual working memory (VWM) can bias the deployment of attention to stimuli in the visual scene possessing the same features. When multiple item representations are simultaneously held in VWM, whether these representations, especially those held in a non-prioritized or accessory status, are able to bias attention, is still controversial. In the present study we adopted an eye tracking technique to shed light on this issue. In particular, we implemented a manipulation aimed at prioritizing one of the VWM representation to an active status, and tested whether attention could be guided by both the prioritized and the accessory representations when they reappeared as distractors in a visual search task. Notably, in Experiment 1, an analysis of first fixation proportion (FFP) revealed that both the prioritized and the accessory representations were able to capture attention suggesting a significant attentional guidance effect. However, such effect was not present in manual response times (RT). Most critically, in Experiment 2, we used a more robust experimental design controlling for different factors that might have played a role in shaping these findings. The results showed evidence for attentional guidance from the accessory representation in both manual RTs and FFPs. Interestingly, FFPs showed a stronger attentional bias for the prioritized representation than for the accessory representation across experiments. The overall findings suggest that multiple VWM representations, even the accessory representation, can simultaneously interact with visual attention. VWM is generally assumed to maintain a target template during visual search, and allows the comparison between the attended items and the target representation3. As a result, the template in VWM will guide attention to the items in the visual scene that are more similar to the target, hence promoting visual search efficiency. In addition, items maintained in VWM and irrelevant to the search target, have also been shown to bias attentional deployment in an involuntary manner. For example, Soto and colleagues8 examined such VWM-based attentional guidance extensively in a line of studies. In their paradigm, participants were required to perform a visual search task during the retaining period of a VWM task. Soto and colleagues found that the item in the visual display matching the features of the memorized item received attentional priority, even when participants knew that this item was always a distractor and hence interfered with the current visual search task. Importantly, memory-driven attentional guidance was a general phenomenon, as it was observed in different situations such as pop-out visual search4, or under high cognitive load conditions5, and with different measures such as standard reaction times9 (RTs) and oculomotor responses (on the first fixation7).The notion that visual working memory (VWM) plays a critical role in visual search is well established and strongly supported by empirical evidence . On the other hand, other studies reported reliable VWM-based guidance by both items using different experimental paradigms such as the rapid serial visual presentation14, manipulations of attentional control settings15, and cueing procedures17.When maintaining two items in VWM, however, the evidence is mixed. On the one hand, some studies reported no significant guidance from two simultaneously maintained items. For example, Peters and colleagues19. When the experimental setting requires participants to memorize two items12, the target template would be necessarily stored in VWM21 with an active or prioritized status18, whereas the other non-target VWM item would become less relevant and would be characterized by an accessory status10. Thus, not only would the target template have priority to guide attention, but this would also result in blocking or even suppressing the guidance from the accessory VWM items22. Since in studies reporting no attentional guidance from the accessory item representation19, one of the items to be held in VWM was specified as target template of the visual search task, it is reasonable to assume that the participants implicitly assigned it an active, high-priority, status, which in turn resulted in weakening the guidance, if any, from the accessory item held in VWM. Recently, in order to clarify this issue, and to constraint the strategy for selective encoding of the items, van Moorselaar et al.23 have firstly asked participants to keep two items in VWM and did not specify either of them as the search template. Then, in order to manipulate the status of VWM representations, prior to the visual search task and the subsequent memory tests, a cue was displayed to indicate which of the VWM items would be asked to be remembered first. According to van Moorselaar et al.23, this procedure would induce participants to prioritize the status of the cued item, and to decreasing the priority of the other to-be-remembered item, thus downgrading it to an accessory status. The results showed that the prioritized VWM item was able to guide attention to the VWM-matched singleton distractor in the visual search display, whereas the accessory VWM item was not. Further, van Moorselaar et al.23 found that without prioritizing one item over the other, none of the items maintained in VWM could bias attention. van Moorselaar et al.23 argued that when multiple representations are simultaneously held in VWM, only the prioritized representation could guide visual attention . In addition, when all VWM items were made equally relevant, both of the items were likely to be assigned an accessory status, which, in turn, could account for the observation that none of them exerted attentional guidance.Some researchers argued that discrepant findings could be attributed to the status of the representations of the items in VWM driven by the task set in the different studies. In this regard, it should be noted that different types of visual search paradigms have been used to investigate this topic. The specific visual search paradigm implemented in a given study can indeed induce the representations of the items held in VWM to assume different priority levels24 have recently reexamined the single-item-template hypothesis with two different types of visual search task. One such task was the singleton-shape search task used by van Moorselaar et al.23 and the other was a gap-location search task that required focal attention to search for the gap location in outline shape stimuli. The results showed significant VWM-based attentional guidance in the gap-location search task but not in the singleton-shape search task when one distractor in the visual search display matched anyone of the two VWM representations . Hollingworth and Beck24 attributed this discrepancy to a possible difference in sensitivity of the two visual search tasks on VWM-based capture. Moreover, they found that when two distractors in the gap-location search task respectively matched the two VWM representations , the guidance was enhanced relative to the “Mem-2 Match-1” condition. These results are not in line with the single-item-template hypothesis and, in contrast, support the conclusion that multiple representations in VWM can simultaneously exert an influence on visual attention , which is referred to as the multiple-item-template hypothesis24.Hollingworth and Beck24, the conclusion that the accessory VWM representation could not guide attention in van Moorselaar et al.23 may be constrained by the type of visual search task used in that study. The present study was aimed at clarifying this issue and to further test whether, when adopting more sensitive visual search tasks, the accessory VWM representation, which is suppressed by the prioritized VWM representation22, can guide attention or not. Whereas Hollingworth and Beck24 were primarily interested in comparing high (2 items) and low (1 item) memory load, here we focused on high memory load in a situation in which the two items were likely to receive a different mnestic priority.In keeping with Hollingworth and Becket al.23, with two crucial modifications. Firstly, the singleton-shape search task was replaced with a tilted-line visual search task similar to that used by Soto et al.26. Since the tilted-line search task was similar to the gap-location search task , this would maximize sensitivity to VWM-based attentional guidance in keeping with Hollingworth and Beck24. Critically, the tilted-line search task has been shown to be effective in detecting VWM-based attentional guidance, at least when participants have to memorize just one item8. The adoption of a visual search task requiring focal attention in the present study was aimed at favoring a closer comparison with other related studies and, more specifically, at clarifying the possible reason underlying the lack of VWM-based guidance from the accessory item reported by van Moorselaar et al.23. Secondly, eye movements were tracked during the visual search task. The link between saccadic eye movements and attention is well established29. Moreover, previous studies have shown that VWM based attentional guidance could occur at early stages during visual search30. For this reason, eye movement dynamics and measures such as the first fixation proportion (FFP) might represent reliable  dependent variables better suited to capture VWM-based guidance of attention. In contrast, manual RTs, which are the standard measure in this specific research topic, represent the behavioral output of a series of cognitive processing stages from perception to response execution. Accordingly, unlike many previous studies, in the present set of experiments we decided to capitalize on eye movements as they represent a more direct, online measure of visual attention. In this regards, it is also worth noting that there is evidence according to which the VWM-based attentional guidance effect as measured in RTs can be weakened or even reversed by cognitive control33. This entails that, compared to FFPs and early oculomotor dynamics, RTs are more prone to cognitive control and therefore more vulnerable and sensitive to strategic factors that might, in principle, affect and mask the phenomenon under investigation34.In order to investigate the attentional guidance from the accessory VWM items, we implemented a variant of the paradigm used by van Moorselaar 24, we expected to observe attentional guidance not only by the prioritized item but also by the accessory item, especially on FFPs. The lack of guidance by the accessory item would instead lend support to the “single-item-template hypothesis”24.In sum, the goal of the present study was to investigate VWM-based guidance of attention focusing on a situation in which two items were to be maintained in VWM with a different mnestic priority. The adoption of a visual search task likely requiring focused attention combined with manual but also eye-movement measures should set the most sensitive experimental conditions to detect VWM-based guidance of attention. Based on the multiple-item-template hypothesis16. Sixteen students  with normal or corrected-to-normal visual acuity and normal color vision took part in this experiment. All were right-handed. An informed consent was obtained at the beginning of the session following a research protocol approved by the Research Ethics Committee of Guangzhou University. All methods used in all experiments performed during the present study were performed in accordance with the relevant guidelines and regulations of the research ethics committee.Sample size was based on previous studies exploring this phenomenon focusing on eye movement measuresThe experiment was conducted in a sound-attenuated and dimly lit room. All visual stimuli were presented with a gray background on a color CRT monitor  about 70 cm away from the participant. Eye movements were recorded from the right eye during the search displays by means of an Eye Link 1000  infrared head-mounted eye tracker . At the beginning of each block the participants performed a standard nine-point grid calibration. An adjustable chin rest helped to maintain head position.The stimuli were drawn by crossing the features of five colors  and five line drawings of shapes . The size of the shapes was approximately 1.9° × 1.9° and the thickness of the border line of the shapes was about 0.5°.et al.23. As shown in Fig. st memory test, and the other was probed by the 2nd memory test. In each memory test, an item appeared at the center of the screen and the participants were asked to judge whether it was the same as the memorized item or not by pressing the “F” and “J” keys of a standard keyboard. “yes” and “no” response trials occurred with the same probability in an unpredictable manner. In the “no” response trials, the item in memory test differed from the memorized item in color, shape, or both features equiprobably.The procedure was similar to that employed by van Moorselaar The visual search display consisted of a target item and a distractor item randomly positioned 6° left and right of a central fixation cross (0.5° × 0.5°). The target item was a colored shape containing a tilted line (38° either to left or to right), and the distractor item was another colored shape containing a vertical line. Both lines were black and with the size of 0.57° × 1.2°. The target and the distractor had no overlapping features. The participants were required to judge the orientation of the tilted line by pressing the “F” key for left-tilted segments and the “J” key for right-tilted segments.st and the 2nd memory test were successively displayed with a separation of 500-ms ISI frame. The participants were instructed to respond accurately to each memory test without time pressure. After the response to the 2nd memory test, a display in the screen was shown to indicate to the participants that they should press the space bar to initiate the next trial.Each trial began with a small white dot (radius = 0.5°) in the center of the screen, which was used for drift correction. Once the eyes of the participants were detected as fixating on the dot for more than 1000 ms, the experimenter immediately pressed the space bar to trigger the appearance of the memory display, which lasted 1000 ms. After a 500-ms Inter Stimulus Interval (ISI), during which only the fixation point was visible, a retrieval cue was presented for 500 ms. After another 500-ms ISI frame, the visual search display appeared, and the participants were required to respond to the target as quickly and accurately as possible. Participants were given a maximum of 2000 ms to respond. As soon as a response was provided or the deadline had expired, the 1st” condition, or the uncued memorized item in the “match 2nd” condition, or did not match any features of the memorized items in the control condition. Each matching type included 72 trials, evenly intermixed within 3 experimental blocks. Before the experimental blocks, each participant had to complete a 20 trial practice phase to get familiar with the experimental task.The distractor in the visual search display might match both color and shape of either the cued memorized item in the “match 1st memory test  was significantly higher than the 2nd memory test , t(15) = 5.17, p = 0.0005). The mean accuracy for the search task was reasonably good  and not analyzed further. The analyses for manual RTs and eye movement data were conducted including only trials with correct responses in both the search task and the memory test task.The mean accuracy for the 1F = 7.35, p = 0.0025, η2p = 0.33. Two-tailed t-tests revealed that the mean RTs for the “match 1st” trials  were significantly greater than RTs for both the “match 2nd” trials  = 3.25, p = 0.005) and the control trials  = 2.79, p = 0.014), whereas the difference between the two latter conditions was not significant (t(15) = 0.72, p = 0.490)  = 17.54, p = 0.0001, η2p = 0.54. Multiple comparisons further revealed that the FFP  was significantly higher than in both the “match 2nd” condition  = 3.82, p = 0.002) and the control condition  = 5.36, p = 0.001). However, in sharp contrast with RT results, FFP in the “match 2nd” condition was also significantly higher than in the control condition (t(15) = 2.19, p = 0.045), suggesting the accessory VWM representation was able to guide attention, although the magnitude of this effect was apparently smaller than that of the prioritized VWM representation.We further estimated the VWM based attentional guidance on FFPs that fell in the region of three matching types of distractors  in the visual search task. FPPs were limited to the first saccadic eye movement time-locked to the onset of the visual search array. In computing the FFPs, we discarded the trials in which (1) the actual first fixations were not located in the region of central fixation ; (2) the first eye movements were less than 100 ms and greater than 500 ms. The repeated-measures ANOVA highlighted a significant main effect of matching type, et al.23 and replicated their RT results, but revealed a different pattern as concerns FFPs. As described earlier, FFPs and RTs have very different time courses, and FFPs  are better suited to reveal the effects on early stages of visual search. Previous researchers have already reported diverging results when comparing FFPs and manual RTs35. The present findings seem to confirm that FFPs are more sensitive to detect the occurrence of attentional guidance as compared to manual RTs.Experiment 1 adopted an experimental procedure similar to that employed by van Moorselaar et al.23 and Experiment 1 of the present study, the retrieval cue was always followed by the visual search task with a fixed temporal interval. This, in turn, might have lead the participants to intentionally prepare for the visual search task before the appearance of the search display. It might be argued that these temporal dynamics could have affected performance in the visual search task. In Experiment 2, we aimed to address this issue by preventing participants to predict the temporal occurrence of the visual search task. This was achieved by presenting either the visual search task or the memory tests after cue offset, in an unpredictable manner  participated in Experiment 2. None of them had taken part in Experiment 1. An informed consent was obtained at the beginning of the session following a research protocol approved by the Research Ethics Committee of Guangzhou University.37. Second, after the retrieval cue and the 500-ms ISI, the participants were presented with either only the visual search task, or only the two successive memory tests. The visual search task and the memory tests occurred with the same frequency and were administered in an unpredictable fashion. As concerns the memory tests, the participants clicked on a standard color wheel38 to report the identity of the color of the memorized items.As shown in Fig. 36, only the average deviation error along the color wheel of the memory tests were computed. These indicated that the memory deviation was greater for the 2nd memory test  than the 1st memory test , t(15) = 9.21, p = 0.0001.The mean accuracy for the search task was 98.47% (SD = 1.8). Considering that the precision of VWM representations has little impact on attentional guidanceF = 3.67, p = 0.039, η2p = 0.21. Two-tailed t-tests showed that mean RTs  and “match 2nd” condition  were nearly identical (t(15) = 0.19, p = 0.85), but both were significantly greater than RTs in the control condition  > 2.23, ps < 0.05). This pattern suggests that regardless of whether sharing common features with either the prioritized or the accessory VWM representation, the distractor captured attention to the same extent.RT data were handled in the same manner as in the previous experiment. The results of the repeated-measures ANOVA for search RTs showed a significant main effect of matching type, see Fig.  for the F = 10.62, p = 0.0004, η2p = 0.43. As shown in Fig. nd” condition  were significantly greater than in the control condition  = 3.08, p = 0.008), but significantly smaller than in the “match 1st” condition  = 2.41, p = 0.031). These results replicated the findings of Experiment 1, indicating that the accessory VWM representation could indeed guide attention although the magnitude of the guidance was smaller than that exerted by the prioritized VWM representation.FFP detection was implemented using the same constraints as in the previous experiment. The results of the ANOVA for FFPs showed a significant main effect of matching type, et al.23 with two relevant modifications. Firstly, we changed the visual search task to enhance sensitivity to detect the VWM-based attentional guidance. Secondly, we tracked eye movements in order to provide a more ecological and sensitive estimate of the attentional guidance effect at early stages of visual search. The FFP results of both Experiment 1 and 2 showed that the accessory VWM representation could indeed guide visual attention, although the magnitude of the guidance effect was smaller than that observed for the prioritized VWM representation. The RT results in Experiment 1 replicated the pattern reported by van Moorselaar et al.23, i.e., no accessory VWM-based guidance was observed. However, when participants were prevented from predicting the appearance of the visual search display and the possibility of verbalizing the memory items was minimized, in Experiment 2, the accessory VWM representation was found to be able to guide attention in the same manner as the prioritized VWM representation also in RT data. Critics may object that not knowing whether the search or the memory test will appear (in Experiment 2) could not only prevent temporal expectancies but also influence memory processes for the two items. Although the different memory measurements of Experiment 1 and Experiment 2 prevent an accurate comparison between the two memory accuracies, it is worth noting that in both experiments the accuracy for the second memorized item was always lower compared to the accuracy for the first item. The observation of a reliable, consistent pattern across the two experiments suggests that the memory processes involved in the two experiments were not strongly dissimilar and this factor did not play a key role in shaping the visual search results.When prioritizing the status of one VWM representation by using a retrieval cue, can the other accessory representations in VWM guide visual attention? The present study addressed this issue by adopting an experimental procedure similar to that used by van Moorselaar st  condition as compared to the control condition (in which no matching features could “push” attention to the opposite location). Analysis of the results in both experiments clearly indicates that visual search performance was always better in the control condition, which, in turn, makes the occurrence of a spatial recoding-like strategy unlikely. According to the VWM-state-hypothesis proposed by Olivers et al.18, the status of VWM representations will affect their interaction with attention, in that the guidance from the accessory representation should be blocked by the prioritized representation in VWM . The present overall results support this view and confirm the impact of the status of VWM representations on attention guidance, in that the prioritized VWM representation had a greater effectiveness in guiding attention as compared to the accessory VWM representations. However, at variance with the predictions of the VWM-state-hypothesis, the present results clearly suggest that the accessory VWM representation can also bias attention, at least to some extent.It could be argued that because in both Experiment 1 and 2 the search array consisted of only two items, this could have led our participants to adopt a spatial recoding-like strategy in that, when a memory-matching distractor was present, this was always counterpredictive with respect to the target location. If this were the case, then the non-matching control condition would be difficult to compare to the two matching conditions. Although we acknowledge this possibility, the overall visual search results of both Experiment 1 and 2 are not consistent with the occurrence of such strategy in our studies. More specifically, had our participants adopted the spatial recoding-like strategy, this should have resulted in a better visual search performance for the match 1et al.23 might refer to the different type of visual search task adopted in the two studies. In the additional singleton task of van Moorselaar et al.23, the attentional guidance is determined on whether the VWM-matched singleton distractor could additionally capture attention. The search mode for this type of task is regarded as the singleton detection mode, and the attentional capture mostly depends on the salience of singleton in search display40. Obviously, the salience of VWM representations is decreased with increasing the number of VWM representation, and this in turn might possibly determine the absence of attentional guidance from the accessory VWM-related singleton in van Moorselaar et al.21 and Olivers et al.30. By contrast, the tilted-line visual search task used in the present study is more likely carried out in a feature-search mode39, as it requires focal attention to search for the target rather than monitoring the emergence of the singleton feature. As a result, such search mode would be less susceptible to the salience of the singleton and even override the attentional capture from the singleton itself41. Following this reasoning, it could be argued that the feature-search mode task is more sensitive for detecting VWM-based attentional guidance as compared to a singleton detection mode task especially when multiple representations ought to be simultaneously maintained in VWM . That said, some researchers13 adopted a visual search task which can be assumed to engage a feature-search mode, and yet did not find evidence for attentional guidance from accessory VWM representations. The possible reason for this discrepancy might be that the complex shapes used in those studies lack the stimulus attributes that can effectively guide attention13 or, alternatively, the accessory VWM representation would lose its competitiveness for guiding attention in the face of the stronger competitor, such as the target template19.One possibility to account for the discrepancy between the present findings and those reported by van Moorselaar 36 have used a different manipulation to induce differences in the status of the to-be-memorized items. After the memory array, their participants were presented with a spatial cue that indicated with an 80% validity the item that will be probed in the memory test. This allowed to distinguish cued and uncued items which, following their rationale, may be interpreted as high-status and low-status items respectively. Overall, no evidence for VWM-based guidance emerged for the uncued items36. It is not easy to directly compare the present results with those of Hollingworth and Hwang36 because in that study visual search performance for the cued items was not assessed. In addition, it might be argued that manipulating expectancies regarding the probed to-be-remembered item might call into play different processes as compared to those involved in the procedure implemented in our studies.Interestingly, Hollingworth and Hwang36 and van Moorselaar et al.23 may result from the different stages of attentional guidance addressed in the measures collected in the different studies. As anticipated earlier, with respect to manual RTs, FFPs can capture attentional guidance at early stages of visual search and are more impervious to cognitive control. Consistent with this interpretation, the present findings show that when considering the different temporal predictability of the visual search display in Experiment 1 and in Experiment 2, the accessory VWM-based attentional guidance is comparatively stable across experiments when assessed by FFPs, but is prone to variation when assessed with manual RTs. Peters et al.22 explored the neural activity of attentional guidance from the target template and accessory representations in VWM, and found that the activity in extrastriate visual cortex was enhanced for target-matching visual input, but was suppressed for accessory-matching visual input, thus suggesting that cognitive control would suppress the interference from irrelevant VWM information to promote the focus on task-relevant information. This interpretation, however, cannot rule out the possibility that attention can still be captured by the accessory-matching visual stimulus before its representation is suppressed. Following this logic, it is possible that the null effect reported on manual RTs in van Moorselaar et al.23 may also be the result of the suppression of the early attentional guidance testified by FFPs in the present experiments.One further important possibility is that the inconsistency between the present results and those reported by both Hollingworth and Hwanget al.18 suggested that only the attentional template in VWM could guide attention, while the other accessory VWM representations would be downgraded to a passive status by the privileged attentional template, and hence could not bias attention. However, Hollingworth and Beck24 and two recent studies by Chen and Feng25 and Bahle, Beck and Hollingworth42 using different types of experimental manipulations and dependent measures have shown that multiple representations in VWM could guide visual attention simultaneously, thus supporting the multiple-item-template hypothesis. Consistent with these findings, the current results also cast evidence for a robust accessory VWM-based attentional guidance on FFPs. These findings suggest that the guidance from accessory VWM representations cannot be completely overridden by the prioritized VWM representation. Additionally, the current results also indicate that the status of the accessory VWM representation is unlikely to be completely downgraded to a passive status, but remains in active status, at least to to some extent.The single-item-template hypothesis of Olivers In conclusion, the present study clearly indicated that multiple VWM representations, irrespective of whether held in either prioritized status or in accessory status, can guide visual attention. In addition, the strength of attentional guidance was greater for the prioritized representation than for the accessory representations. This latter evidence was observed in both experiments and future studies will need to address whether this pattern can be observed across other types of experimental manipulations and settings, in order to provide indications as to whether VWM-based guidance of attention could go beyond a simple all-or-none dichotomy and reflect a more nuanced prioritization hierarchy.


Flammulina velutipes has been recognized as a useful basidiomycete with nutritional and medicinal values. Ergosterol, one of the main sterols of F. velutipes is an important precursor of novel anticancer and anti-HIV drugs. Therefore, many studies have focused on the biosynthesis of ergosterol and have attempted to upregulate its content in multiple organisms. Great progress has been made in understanding the regulation of ergosterol biosynthesis in Saccharomyces cerevisiae. However, this molecular mechanism in F. velutipes remains largely uncharacterized.F. velutipes (three replicate sets for each stage), were sequenced using the Illumina HiSeq™ 4000 platform, resulting in at least 6.63 Gb of clean reads from each library. We studied the changes in genes and metabolites in the ergosterol biosynthesis pathway of F. velutipes during the development of fruiting bodies. A total of 13 genes (6 upregulated and 7 downregulated) were differentially expressed during the development from mycelia to young fruiting bodies (T1), while only 1 gene (1 downregulated) was differentially expressed during the development from young fruiting bodies to mature fruiting bodies (T2). A total of 7 metabolites (3 increased and 4 reduced) were found to have changed in content during T1, and 4 metabolites (4 increased) were found to be different during T2. A conjoint analysis of the genome-wide connection network revealed that the metabolites that were more likely to be regulated were primarily in the post-squalene pathway.In this study, nine cDNA libraries, prepared from mycelia, young fruiting bodies and mature fruiting bodies of F. velutipes.This study provides useful information for understanding the regulation of ergosterol biosynthesis and the regulatory relationship between metabolites and genes in the ergosterol biosynthesis pathway during the development of fruiting bodies in  Flammulina velutipes (F. velutipes) has been recognized as a model industrial basidiomycete; it is one of the most commonly used edible fungi, serving as an excellent source of vitamins, amino acids, polysaccharides, fibre, terpenoids, phenolic acids, steroids, fatty acids and other metabolites, and is widely cultivated worldwide [F. velutipes, including anti-inflammatory and immunomodulatory proteins [F. velutipes was 35.5 mg/100 g in wet weight or 68.0 mg/100 g in dry weight [Edible fungi are the sixth largest crop in China with a total output of 33 million tons in 2015 [proteins , antitumproteins , antimicproteins , and antproteins , 10. Theproteins , 12. They weight , 14.28H43OH) is a typical fugal sterol and an important constituent of various membrane structures of fungal cells, and it contributes to multiple physiological functions in cells, such as cell viability, membrane permeability, membrane fluidity, membrane integrity and intracellular transport. Therefore, when ergosterol is lacking, abnormal cell membrane function and even cell rupture may occur [Ergosterol  are transcription factors that bind to the sterol regulatory element DNA sequence. Therefore, the manipulation of biosynthesis genes by genetic engineering may be an effective way to modulate sterol biosynthesis and intracellular sterol components. Although progress has been made in the metabolic and genetic engineering of synthetic pathways in Saccharomyces cerevisiae (S. cerevisiae), the roles of ergosterol biosynthesis genes in fruiting body growth and associated metabolic changes remain a mystery.Ergosterol and its derivatives are obtained mainly by chemical synthesis, genetic engineering and metabolic engineering . BecauseERG7, ERG11, ERG24, ERG25, ERG26, ERG27, ERG6, ERG2, ERG3, ERG5 and ERG4 [The ergosterol biosynthetic pathway can be divided into two parts: the mevalonate pathway and the post-squalene pathway Fig. . Part 1 F. velutipes samples from three developmental stages: the mycelia stage (FrI), the young fruiting bodies stage (FrII) and the mature fruiting bodies stage (FrIII). The transcriptome technique was used to identify changes in the expression of genes involved in the ergosterol biosynthesis pathway during fruiting body development. Thereafter, the metabolites in this pathway were completely scanned by nontargeted metabolomic techniques. We explored the regulatory relationship between genes and ergosterol biosynthesis during fruiting body development. The results had vital significance for understanding the metabolic pathway of ergosterol biosynthesis in F. velutipes.Multi-omics has become a common biological approach for systematic genome analyses , 29. In F. velutipes at three different developmental stages were prepared and sequenced using the Illumina HiSeq™ 4000 platform  from A total of 4907 (2798 downregulated and 2109 upregulated) DEGs were identified during the first developmental transition (T1), and 1383 (551 downregulated and 832 upregulated) DEGs were identified during the second developmental transition (T2)   to observe differences in metabolic constituents among the three developmental stages of F. velutipes. The metabolome used the VIP values of the first two principal components of the multivariate PLS-DA model and a combined univariate analysis of fold change and p-value to screen for differentially expressed metabolites. The screening conditions are as follows: 1) VIP ≥ 1; 2) fold change ≥1.2 or ≤ 0.83 and 3) p-value < 0.05. These three factors were taken into account to obtain a common ion. Metabolic pathway analysis was based on the KEGG database. To compare the metabolic constituents in the three developmental stages, datasets obtained from UPLC-TOF-MS in the ESI+ (ESI−) mode were subjected to PCA. The results showed different metabolic profiles among the three groups  and PC1 in ESI− (42.56%) were clearly separated between the FrI and FrII groups. The differences between the FrII and FrIII groups resulted from PC2 (15.45% variables) in ESI+ mode and PC2 (16.69%) in ESI− mode. A total of 1742 (2154) and 751 (944) mass ions were selected between the FrI and FrII groups and between the FrII and FrIII groups in the ESI+ (ESI−) mode, respectively  accumulated in mature fruiting bodies. In early fruiting body development, the accumulation of metabolites greatly contributes to the acquisition of fruiting traits [F. velutipes, PDA, MCM and sawdust) [F. velutipes.In this study, through metabonomic analysis, we found that metabolite profiles were significantly different and that the contents of ergosterol biosynthesis-related metabolites significantly changed among the three developmental stages Tables  and 4. Tg traits . In mostg traits , 33. In sawdust) . HoweverERG10 gene encodes an acetoacetyl-CoA thiolase that catalyses the formation of acetoacetyl-CoA from two acetyl-CoA molecules. When the levels of some sterols in the cell are low, the ERG10 gene is expressed at a higher level and then regulates the activation pathway [ERG10 was downregulated during T1, and the results indicated that the gene may be subject to feedback regulation by sterols. In previous studies, ERG1 was identified as the key regulator of post-squalene biosynthesis in S. cerevisiae and Trichoderma harzianum [ERG1 could significantly increase ergosterol biosynthesis [S. cerevisiae, the deletion of ERG26 is lethal and disrupts the synthesis of ergosterol [ERG26 is essential for cell growth and impacts the synthesis of ergosterol. The various enzymes in the ergosterol biosynthesis pathway cooperate to tightly regulate the ergosterol content. Moreover, the genetic engineering of F. velutipes has been very successful [The  pathway . In thisarzianum , 36. Forynthesis . In S. cgosterol , 39. Theccessful , 41. GenS. cerevisiae has been well characterized, few efforts have been made to examine ergosterol biosynthesis in F. velutipes [Effective genetic engineering approaches for efficient ergosterol production from the mycelia or fruiting bodies of a fungus cannot be devised until the metabolic pathway and regulation mechanism are well understood. Although the biosynthesis pathway of ergosterol in elutipes . The reselutipes . A totalUPC2 has been reported to upregulate target genes involved in the biosynthesis of sterols by activating the sterol response elements in their promoter regions [Candida albicans ERGs . Our results revealed that the expression levels of most of these genes differed, which was consistent with the regulatory effect of UP2C on these genes. Most of these genes were found to be related to the post-squalene pathway, which is promising for the improvement of sterol biosynthesis in F. velutipes [The efficiency of ergosterol biosynthesis is determined by rate-limiting enzymes, and more crucially by the optimal coordination of all enzymes . The tra regions , 44. Theelutipes . The reselutipes . In summF. velutipes.The in vivo biosynthesis of ergosterol is a complex metabolic process involving a variety of enzymes (at least 20), reactions and genes. Due to the different positions of each reaction in the metabolic pathway, the role of metabolic regulation is different, and therefore the effects of high expression of related genes are not the same. Some genes function well in ergosterol synthesis when properly expressed, while the overexpression of these genes may inhibit ergosterol biosynthesis. These results will shed light on the molecular mechanisms responsible for ergosterol biosynthesis in F. velutipes. Correlation analysis highlighted regulatory genes closely associated with individual metabolites or much larger networks of genes and metabolites, thereby suggesting that a strategy based on the combined analysis of different developmental stages can be very helpful for pinpointing candidate regulatory genes linked to compositional changes and fruiting body development in F. velutipes. Researchers could utilize this dataset in genetic approaches to clarify the mechanism of ergosterol regulation. In summary, this study will be instrumental for further research on the biosynthesis of sterol-related metabolites in F. velutipes.This study explored the regulatory relationship between genes and ergosterol biosynthesis by jointly analysing transcriptional and metabolic changes at three developmental stages of F. velutipes samples from three different developmental stages, the mycelia stage (FrI), the young fruiting bodies stage (FrII) and the mature fruiting bodies stage (FrIII), as experimental materials [F. velutipes mycelia were grown on potato dextrose agar (PDA)  plates at 23–25 °C in darkness [F. velutipes samples at three developmental stages were collected under sterile conditions, immediately frozen and stored in liquid nitrogen at − 80 °C before the metabolic content determination and RNA isolation.This article used aterials , 29, 47.darkness . For theF. velutipes (three replicates of each stage) were sequenced by using the Illumina HiSeq™ 4000 platform.Total RNA was extracted with the RNAprep Pure Plant Kit . The total RNA concentration, RIN value, 28S/18S and fragment size were determined using an Agilent 2100 Bioanalyser  with the Agilent RNA 6000 Nano Kit. The purity of the samples was measured using a NanoDrop™ ultraviolet spectrophotometer. After the isolation and fragmentation of total RNA, eukaryotic mRNA was enriched by using Oligo (dT) coupled to magnetic beads. In this work, nine cDNA libraries produced from the three stages of F. velutipes KACC42780 was downloaded from the NCBI under accession number PRJNA191921. GeneMark-ES was used to predict genes from the genome sequences [The reference genome of equences . The CDSequences . The funequences .2(fold change) ≥ 1 and a false discovery rate (FDR) < 0.01 were identified as a DEG.Reads were first processed using in-house Perl scripts and filtered to remove low-quality sequences, contaminated adapters and poly-N sequences. The raw reads were mapped to the reference genome  using Hihttp://www.uniprot.org/uniprot) database. Kyoto Encyclopedia of Genes and Genomes (KEGG) pathway and clusters of orthologous groups (COG) annotation was performed using Blastall software against the KEGG database and the COG database, respectively, with a cut-off E value of 1e− 5. For functional enrichment analysis, GO terms of DEGs were compared to the genome background, and the corrected p-value less than 0.05 was set to judge the significantly enriched GO terms. The pathway enrichment analysis was performed similarly using the KEGG database, and a p-value less than 0.05 was the threshold.The protein sequences of DEGs were used to perform functional prediction and classification using Eggnog . Functio−△△Ct method.To validate internal control genes for expression analysis, the ABI StepOnePlusTM RT-PCR System  was used to perform RT-qPCR and analyse the DEGs related to ergosterol biosynthesis. Amplification primers were designed using GenScript Real-time PCR (TaqMan) Primer Design, and the primer sequences are provided in Additional file F. velutipes at three developmental stages  were tested by using UPLC-MS/MS. All samples stored at − 80 °C were placed in a freezer at − 20 °C for 30 min and then thawed at 4 °C. A 25 mg tissue sample was weighed, and placed in an EP tube, 800 μl of a cooled solution of methanol/water (1:1) was added to each EP tube, and two small steel balls were frozen and placed in each EP tube. The sample was then placed in a tissue lyser and the parameters were set to 35 Hz for 4 min. After grinding, the ball was removed and the tube was placed in a − 20 °C refrigerator for 2 h. The sample was centrifuged at 30,000×g for 15 min at 4 °C. The EP tube was carefully removed from the centrifuge, and 550 μl of each sample was transferred to a new EP tube. The sample was placed in the rack of a centrifuge, and an image was obtained according to the order of the samples on the task sheet. The samples were subsequently analysed by LC-MS, and the remaining original samples were provided to the sample manager for storage.In this study, 18 samples produced from All samples were produced and processed following protocols for LC-MS data analysis. First, chromatographic isolation was performed by using a UPLC system . Default settings were adopted for the column type, column temperature, mobile phase flow rate, mobile phase solvent ratio and other program settings . The injTo unveil the regulatory mechanism of ergosterol biosynthesis, we measured the associations between metabolome and transcriptome data by calculating the cosine coefficients between fold changes of the metabolite-gene pairs. For this purpose, the mean of all biological replicates from each stage in the metabolome data and the mean value of expression of each transcript in the transcriptome data were calculated. The fold changes in developmental transitions were calculated for both the metabolome and transcriptome data. Finally, the correlation matrix was imported into Cytoscape software to visualize the gene-metabolite network .F statistics and the James-Stein estimator to calculate the error variance and selected genes (fold change ≥2 and FDR < 0.01) with significant differential expression. Principal component analysis (PCA) of data from F. velutipes  was performed to observe differences in metabolic composition and expressed genes from the three developmental stages. The gene expression data and metabolite data were standardized as Z scores. Pearson’s correlation coefficient was calculated both among the gene expression data and between the gene expression and metabolite accumulation data. For correlations with p < 0.01, correlation matrices were visualized using heat maps generated with MultiExperiment Viewer software version 4.0 [p value was calculated using an independent two-sample t-test.We used sion 4.0 . The p vAdditional file 1: Table S1. The statistics of RNA-Seq data at different developmental stages of F. velutipes. Figure S1. The DEG statistics at different stages of F. velutipes. Figure S2. GO functional annotation and classification of DEGs during T1 of F. velutipes. Figure S3. GO functional annotation and classification of DEGs during T2 of F. velutipes. Figure S4. Differential expression profiles of nine DEGs at different developmental stages of F. velutipes. GAPDH and β-actin were used as internal controls. There were three biological replicates and three technical replicates for each gene. Table S2. The results of differential ions identification at different developmental stages of F. velutipes. Table S3. Identified metabolites in the ergosterol biosynthesis during T1 of F. velutipes. Table S4. Identified metabolites in the ergosterol biosynthesis during T2 of F. velutipes. Figure S5. Verification results of the end product ergosterol in metabolomics. (A) LC-MS spectrum of the ergosterol standard. (B) LC-MS spectrum of ergosterol from F. velutipes. Their corresponding m/z and retention time (RT) are indicated in the figure. Excel S1. The profiles of metabolites and gene expression at three different developmental stages of F. velutipes using Pearson’s correlation coefficient. Excel S2. Network analysis results of metabolites and genome-wide genes at three different developmental stages of F. velutipes by Cytoscape. Table S5. Primer sequences of DEGs related to ergosterol biosynthesis in F. velutipes.


The retina is the light sensitive part of the eye and nervous tissue that have been used extensively to characterize the function of the central nervous system. The retina has a central position both in fundamental biology and in the physiopathology of neurodegenerative diseases. We address the contribution of functional genomics to the understanding of retinal biology by reviewing key events in their historical perspective as an introduction to major findings that were obtained through the study of the retina using genomics, transcriptomics and proteomics. We illustrate our purpose by showing that most of the genes of interest for retinal development and those involved in inherited retinal degenerations have a restricted expression to the retina and most particularly to photoreceptors cells. We show that the exponential growth of data generated by functional genomics is a future challenge not only in terms of storage but also in terms of accessibility to the scientific community of retinal biologists in the future. Finally, we emphasize on novel perspectives that emerge from the development of redox-proteomics, the new frontier in retinal biology. The vertebrate retina is composed of three layers of neurons, two layers of synapses and Müller glial cells which stretch radially across the thickness of the retina . The outThe eye and its photosensitive component, the retina, have fascinated evolutionary biologists for more than a century. The eye was perceived as a challenge to the theory of evolution due to its extreme perfection and complexity. Charles Darwin wrote that it is inconceivable to our imagination that the formation of a structure as perfect and complex as the eye could result from natural selection even if this may have been really achieved through numerous inherited gradations useful to animals . Some auHydra vulgaris use for capturing prey, is regulated by light and opsin-mediated phototransduction [The evolution of vision can be traced back to its putative origins in cyanobacteria a prokaryotic eubacteria found in the oldest known fossils on Earth, three and a half billions years ago. Cyanobacteria can sense the light–dark cycle and maintain oscillatory behaviors even under constant environmental conditions. This timing mechanism, called the circadian clock, involves in eukaryotes biochemical oscillators and multiple intertwined transcription/translation feedback loops. Cyanobacteria respond to light by phototactic motility behavior, a movement of a whole organism toward the light, or inversely avoidance of light . Interessduction . Other csduction . It is nsduction . The preSon of sevenless (SOS) was identified based of the ability of SOS alleles to suppress the eye phenotype of the sevenless mutant that carries an inactive receptor tyrosine kinase gene. Sevenless is essential for R7 photoreceptor differentiation [The power of drosophila genetics combined with microscopic examination of the phenotype of the drosophila eye has formed the basis of very powerful genetic screens. A block in photoreceptor cell differentiation triggers the roughening of the external eye surface . Son of ntiation . SOS wasntiation .RB1) was the first tumor suppressor gene identified [An integrated model of retinal cell-fate in the vertebrate retina was obtained using in vivo lineage tracing using retroviral vectors and autoradiographic tracers ,19,20. Nentified ,33,34. Tentified .+ as a co-factor to deacetylate acetyl lysine residues of protein substrates, among which histones. Consequently, epigenetic control is tributary to the metabolism of the cell since NAD+ is produced among other sources by the reduction of pyruvate to lactate by lactate dehydrogenase A [During the construction of the retina A,B, trangenase A . This isgenase A .https://sph.uth.edu/retnet/). Among the most common forms of such diseases, retinitis pigmentosa affects nearly 2 million people worldwide [A certificate of authenticity of the genes regulating retinal developmental is indirectly provided by their implication in inherited retinal dystrophies ,50,51. Iorldwide ,55. In rorldwide . On the orldwide . Cone roorldwide . Stargarorldwide . Leber corldwide ,61. Patiorldwide ,63,64. Torldwide . Within orldwide ,67. Bardorldwide ,69. The orldwide .rd1) mouse [RHO) from those expressed by rods and cones . Forty-eight percent of the genes examined from surgical specimens of human retinal detachment with both rod and cone death (PR-death), displays a similar rod-like expression pattern, confirming what observed in the rd1 retina. We next considered that some of the rare genes are actually not carrying disease-causing mutations. We excluded genes with rare alleles, leaving us with 45 genes (50%) with a report of frequency of mutations in retinitis pigmentosa. Those 45 genes account for 53% of autosomal dominant, 45% autosomal recessive and 66% X-linked RP. Twenty-nine out of 40 genes whose profile could be examined, rod-like expression patterns account for 73%. Altogether and as expected, the listed genes carrying mutations causing retinitis pigmentosa is biased toward genes expressed by photoreceptors (rods and cones). Virtually all genes involved in the phototransduction cascade and in the vitamin A cycle carry mutations in individual patients suffering of inherited retinal degenerations [RPE65 gene was identified as a gene encoding for a protein of 65 kDa expressed specifically by the retinal pigmented epithelium [The genetics of inherited retinal degeneration is very complex as illustrated here by the case of retinitis pigmentosa (RP) . We exam1) mouse  as well 1) mouse , the mou1) mouse . Among terations ,75. Conserations ,79,80,81ithelium . TherebyHomo species and their migrations in prehistoric times [Many of the retinal genes of interest described above were identified in the pre-genomic times, but all the scientific community of biologists were eager to get the DNA script of the human and mouse genomes to facilitate their understanding of any biological scenarios under their scrutiny. Sequencing the entire human genome was a tedious task and its achievement has been compared to man’s first steps on the moon fifty years ago. Once the technological barrier had been overcome, the sequencing of other genomes became greatly facilitated ,219. Comic times ,226,227.ic times . Comparaic times ,232,233.ic times ,235.19 base pairs (bp), to which is added 4.15 × 1024 bp of the human microbiome. Biological sciences have an asymptotically reaching problem similar to that of the most powerful particle accelerator, the large hadron collider (LHC) of the European Council for Nuclear Research (CERN). LHC manages to store the data using the grid built on the technology of the World Wide Web (invented at CERN in 1989). Collisions in the LHC produce too much data to record, so they are filtered to retain only the interesting ones for analysis. Will this filtering be ethic for genomic data? This is an eminently complex question that will not be resolved here.The exponential increase in the amount of data generated by genome sequencing has given birth to bioinformatics, later on to systems biology and nowadays to the concept of big data. The size of the genome of the entire human population is 2.42 × 10CFH) gene on chromosome 1q32 have been associated with an increased risk for AMD [CFH gene focused on the Y402H coding variant that alters a single amino acid in the CFH protein, but additional SNPs shows higher risk [ARMS2/HTRA1, it has not yet been possible to determine if the AMD susceptibility results from the variants in the ARMS2 or the nearby HTRA1 gene or both [http://amdgenetics.org/). IAMDGC performed a meta-analysis of the results of 14 GWAS representing > 17,100 advanced AMD cases and >60,000 controls of European and Asian ancestry. The meta-analysis examined 2,859,744 imputed and genotyped SNPs [−8. Among these loci, 12 were previously associated with AMD, whereas seven were newly identified by the consortium [SCL16A8, distributed across 34 loci were identified [The retina is not in rest in this revolution. Age related macular degeneration (AMD) affects a region of the retina, the macula that is not present in non-primate species, and consequently a rodent model of AMD is still missing. This led researchers to concentrate their efforts on the genetic predisposition of AMD. AMD is a polygenic and polyfactorial disease as opposed to Mendelian inherited retinal degenerations, such as retinitis pigmentosa. Family and twin studies have demonstrated that the susceptibility for AMD is under the influence of the genome. Age and a positive family history of AMD are the two strongest risk factors for AMD. It has been shown that an individual with a sibling or a parent with AMD is 12–27 times more susceptible than someone from the general population to develop AMD. Geneticists have successfully employed the genetic variations in the sequence of the human genome among individuals to map loci that may contain genes carrying risk alleles for polygenic diseases . Single  for AMD ,240,241.her risk . For the or both ,244. A mped SNPs . This mansortium . The knoentified ,248. Oveentified . The therd1 retina [rd1 mouse [As illustrated above by examples in the field of research on the retina, both developmental biologists and human geneticists have a strong interest in genes with retina- and photoreceptor-restricted expression profiles. The selection is generally achieved by comparing global gene expression between specimens that represent two extreme conditions. This was originally done by subtractive and differential cloning using the degenerated 1 retina . The con1 retina ,252. Rap1 retina ,254,255.1 retina  ArrayExp1 retina  and the 1 retina . The US 1 retina ,260. Thed1 mouse , retinald1 mouse  or hypoxd1 mouse , models d1 mouse ,264 or bd1 mouse ,265, or d1 mouse . A modeld1 mouse . The outd1 mouse ,269. Retd1 mouse ,271. Thed1 mouse .RHO). This restriction led other researchers to develop a broader approach using engineered miRNA, or small interfering RNA (siRNA) to suppress the expression of any dominant mutations in the rhodopsin gene by RNA interference and to replace the normal copy of that gene by its reintroduction using the same vector [Soon after, retinal biologists got interested in gene expression in terms of alternative splicing because this phenomenon alters dramatically the function of the encoded proteins . The mote vector .Biologists working on retinal development are constantly looking for cell specific markers that can be used to specify retinal cell fate during development to reveal its transcriptional code . VariousThe assembly of genomes and their annotation allow the in silico translation of putative messenger RNA into proteins and deduce their molecular weight. By digesting a complex mixture of proteins by an endoproteinase, as trypsin, the individual mass of each small peptide of this collection can be accurately measured by mass spectrometry. By comparing the masses of these tryptic peptides with the in silico-digested proteins from the genome you can identify proteins in any protein mixture, even of proteins that have not yet been characterized ,293. BioBeside this repertoire of proteins expressed in the eye, differential proteomic analysis was quite successfully applied to the physiopathology of inherited retinal, degenerations by comparing disease specimens to healthy ones ,299. TheInitially, proteomics was mainly semi-quantitative, the results were pointing to proteins that were more abundant in one of two groups of specimens by differential analysis, but the need for quantitative data was pressing. Diverse protocols were developed with the use of non-radioactive isotopes that allows the discrimination of tryptic peptides of identical sequences by the difference in their mass. One such approach relies on the incorporation of these isotopes in cell culture as the stable isotope labeling by amino acids in cell culture (SILAC) ,318. AnoProteomics, which is a technology that does not use an amplification step as polymerase chain reaction (PCR), captures the most abundant protein of the cells, among which are the enzymes of the cellular metabolism. Metabolomics is an approach that measures the concentration of metabolites in a given specimen and was also applied to retinal specimens to decipher their metabolic status ,324,325.It is certainly thanks to the fact that mass spectrometry is capable of identifying unambiguously most of the protein content of a protein fraction of interest that proteomics became widely used by retinal biologists to identify protein-protein interactions. The diverse methods used to purify these fractions rely mostly on the question addressed. The use of an antibodies against the bait protein is an appropriate choice for co-immunoprecipitation ,332,333 3+, Ga3+, and Al3+) and this interaction makes it possible to enrich phosphorylated peptides from rather complex peptide samples. Phosphoproteomics is still a method that is more time consuming than the use of antibodies raised against phosphopeptides, but it is much more precise.Since it is clear to all biologists that cell signaling is regulated at the post-translational level, researchers got interested by post-translational modifications . The comAnother type of post-translational modifications is generated by reactive oxygen species (ROS). Due to the probable involvement of ROS in neurodegenerations, which include the inherited retinal degenerations and aging related diseases, such as AMD, a plethora of studies relating damages of oxidation on macromolecules in the pathology of the retina have been published. Retinal proteins can be modified irreversibly by 4-hydroxynonenal, a product of lipid peroxidation . CarboxyRetinal biologists are now immerged in the post-genomics area. Questions concerning the development of the retina and its physiopathology are becoming more and more complex and the results of each investigation generally contain data that is far more complete than what was the focus of each study. It is of great interest to the community of biologists to keep track of these data that could be analyzed posteriorly by experts in their respective field. This objective is not trivial due to the exponential growth of the volume of data generated by a single functional genomics experiment. Biology is being confronted by the same trend than is also challenging the society: the increase in the amount of information is not proportional to our understanding of it. Nevertheless, functional genomics is a great leap forward to the future.