But these simulated experiments do not account for individual variations and are difficult to investigate when the listener is wearing hearing devices. Hence, the free-field systems are ideal for spatial hearing experiments. However, these systems are not readily available and must be customized based on experimental needs. This paper provides a brief overview of a spatial hearing research facility that is customized to perform experiments on individuals with normal hearing and hearing aid users.   Specifications tableSpatial hearing is the ability of the human auditory system to utilize the information concerning the location of the sound source and the sound arrival path for the interpretation of the sound. Auditory localization, an underpinning mechanism of spatial hearing not only assists in orienting to the sound source but also in perceptually segregating the target sound and the interfering noise arriving from different directions Though spatial hearing is a free field phenomenon, the revolution in binaural audio synthesis techniques has enabled researchers to assess spatial hearing abilities under headphones The current lab is housed in a rectangular spaced room about 6.7 × 5 × 2.3 m (length x width x height). The floors and ceiling are constructed using concrete, while bricks are used to construct the walls. The adjacent blocks on either side of the lab have classrooms and no heavy noise generators or sound sources are present around the lab. To avoid background noise, the water and drainpipes were not allowed to pass through the lab.To avoid participant distraction during the experiments, the lab has been divided into an experiment room (4.8 × 5 × 2.3 m) and a control room (1.8 × 5 × 2.3 m). The partition between the experimental and control room has a glass window for direct visual monitoring and an acoustically treated door. The personal computer and soundcards are installed in the control room to reduce the background noise. Instead of personal cable bays, we used universal cable ducts to connect loudspeakers and soundcards, which enable us to modify loudspeaker arrangements with minimal effort .Fig. 1ThThe experimental room is acoustically treated. The ambient noise and reverberation time were measured using the M4261 microphone of NTi Audio XL2 sound level meter, positioned at the center of the experimental room. The ambient noise was measured following American National Standard Institute standards The reverberation time (RT60) is defined as the duration required for the intensity to drop by 60 dB after the sound source has stopped Thirty-six speakers were positioned circularly on a custom-made plywood ring frame with a width of 8.6 inches and a thickness of 2 inches. The circular frame has a diameter of 3.75 m and a circumference of 11.78 m. It was covered with a layer of an acoustic damper to reduce vibrations and floor-mounted with 12 cylindrical steel rods of identical height, thereby giving the effective height of 1 m to the ring frame. f = c/λ where c ≈ 344 m/s and λ = 2 × 1.87 m). However, this distance may be slightly greater than the critical distance, where the reflected signal may dominate over the direct signal The placement of the participant is determined by considering the far-field hearing phenomenon wherein the ITD and ILD cues remain constant even with an increase in the distance. When the distance between the loudspeaker and the participant reduces below 1 m, the ILD increases, especially, when the loudspeakers are placed in the lateral position. Nevertheless, the ITD cues remain constant irrespective of the distance Placing the participants beyond the critical distance has some advantages as well, especially, when measuring the hearing aid benefit using spatial release from masking. Positioning the participant beyond the critical distance may help to simulate a few real-life situations where the reflected sound is stronger than the direct sound The requirement for equipment with specific characteristics is determined by the experiments done in the lab. In general, for spatial hearing experiments, compact speakers were advised because they can allow us to fit more speakers into a small space and significantly reduce the acoustic reflections produced by the speakers. Often the compact studio monitor speakers are preferred for localization experiments. Most commercially available studio monitor speakers use multiple drivers to produce low and high frequencies separately. The cross-over determines the range of frequencies to be reproduced by each driver. However, at the cross-over point, some frequencies would be reproduced by both drivers leading to unpredictable phase distortions. Also, the multiple drivers in a speaker will act as different points of sources, introducing undesirable localization cues. Phase distortion at the cross-over is not a major factor for the experiments insensitive to phase. Also, the problem of multiple sound sources can be overcome by positioning the speakers far or filtering the input signal. On the other hand, single driver speakers are free of cross-over and multiple source effects. However, the single driver speakers do not have a flat frequency response whereas speakers with multiple drivers produce a relatively flat frequency response.In the current setup both single and multiple driver speakers are used. Thirty-six active loudspeakers including 24 Avantone Mixcube  and 12 Yamaha MSP5  were installed in the current setup. Avantone Mixcube is a single driver speaker whereas the Yamaha MSP5 is a two-way speaker having a woofer and tweeter to reproduce low and high frequencies respectively. The sensitivity of the speakers is adequate to perform unaided listening experiments in individuals having up to a moderately severe degree of hearing loss and aided listening experiments up to a profound degree of hearing loss. The technical characteristics of Avantone and Yamaha studio monitors were represented in Experiments involving two-way speakers will be carefully designed by considering their crossover frequency. The crossover frequency represents the frequency at which the transition occurs between the drivers, which is 2.5 kHz for Yamaha MSP5. So, these speakers will be used to deliver either low or high-pass filtered signals with a cutoff frequency of 2.5 kHz. This allows only one driver of the speaker to be active at any given time, thereby overcoming the phase distortions. The high-frequency driver of Yamaha MSP5 is positioned at a height above the total height of Avantone Mixcube which would act as a source cue. Therefore, these drivers are kept unused when used along with single driver speakers. These modifications in loudspeaker arrangement can be done with minimal effort since our lab uses XLR cables with universal cable ducts for all the speakers.Typically, the single driver speakers do not have a flat frequency response, and their sensitivity and frequency response might drift over time depending on their usage. As a result, it needs to be equalized across the speakers in terms of both sensitivity and frequency regularly to avoid their potential contribution to measured performance. For this objective, a digital equalization filter was designed using Gaussian white noise  delivered through loudspeakers and recorded using an omnidirectional microphone (M4261) placed at the subject position.The equalization was done in two steps. First, the intensity across the speakers was equalized digitally in MATLAB. Second, the magnitude equalization was performed using a 1024th order Finite Impulse Response (FIR) filter. The filter coefficients were saved for each speaker, which was used to equalize the speaker outputs during experiments. The filtering of signals during equalization attenuated the overall intensity of the signal, hence the RMS amplitude of processed signals was equalized. As a result, we obtained 91.4 dBA and 90.2 dBA for the Avantone Mixcube and Yamaha MSP5 respectively, while maintaining a flat frequency response of ± 2 dB between 100 and 10,000 Hz , which are housed in the control room. Both audio interfaces MOTU 24Ao and MOTU 16A can deliver balanced outputs up to 96 kHz sampling rates. Since we use two audio interfaces, they will operate on different digital clock settings resulting in output delays. We have synchronized the digital clock of MOTU 24Ao with MOTU 16A using a Cat-6 Ethernet cable. Alternately, the audio interfaces can be connected to a local network via MOTU AVB switch for precise control over the clocking and latency optimization. MOTU AVB switch streams the audio signals at 1 Gbps with minimal network latency and provides a unified digital clock that allows synchronization in nanosecond precision.In our lab, we use the Digital Signal Processing toolbox of MATLAB  to design spatial hearing experiments on a Lenovo P340 workstation with an Intel Xeon W-1250 (3.3/4.7 GHz clock speed and 12 MB cache) and 64 GB non-ECC RAM. The subject responses can be acquired either by using a manually controlled response application designed using MATLAB application designer or by recording the verbal responses using a wireless microphone. For playback, the ASIO4ALL sound driver is chosen over the default windows driver, since the former is more efficient to handle multi-channel audio signals and enables a connection of the audio interfaces directly to the software thereby reducing the overall output latency. To implement EEG recordings in the future, we installed a computer with such computational specifications. Currently, it is sufficient to generate and deliver multi-channel audio signals as well as apply equalization filters on the fly without any disturbances.The spatial hearing lab setup is currently used to investigate spatial hearing acuity using the RMS localization error metric and spatial release from masking (SRM). The setup enables RMS error and SRM measurements in unaided listening conditions in individuals with a moderately severe degree of hearing loss or less. Nevertheless, aided listening experiments can be performed up to the profound degree of hearing loss.


Temporal epilepsy, one of the most common presentation of this pathology, causes excessive electrical discharges in the area where we have the final station of the auditory pathway. Both the anatomical and functional integrity of the auditory pathway structures are essential for the correct processing of auditory stimuli.to check the Auditory Processing in patients with temporal lobe epilepsy regarding the auditory mechanisms of discrimination from sequential sounds and tone patterns, discrimination of the sound source direction and selective attention to verbal and nonverbal sounds.eight individuals with temporal lobe epilepsy were assessed, after excluding those with non-confirmed diagnosis or with the focus of discharges not limited to this lobe. The evaluation was carried out through special auditory tests: Sound Localization Test, Duration Pattern Test, Digits Dichotic Test and Non-Verbal Dichotic Test. Their performances were compared to the performances of individuals without neurological diseases (case-control study).similar performances were observed between patients with temporal lobe epilepsy and the control group regarding the auditory mechanism of sound source direction discrimination. Comparing the other auditory mechanisms assessed, the patients with temporal lobe epilepsy presented worse results.individuals with temporal lobe epilepsy had more deficits in auditory processing than those without cortical damage. Epilepsy is a set of clinical manifestations that may reflect a temporary neural dysfunction - abnormal and excessive electric dischargesThe auditory pathway ends at the temporal lobe (primary and secondary auditory cortex), after going through many central and peripheral structures of the auditory system. Knowing that for a correct analysis and interpretation of the information received through one's hearing (auditory processing) it is necessary to have full anatomical and functional integrity of all these structures and that the electric discharges caused by the crisis may cause neuronal losses in the region where they occur, therefore it has been thought that there may be difficulties in the mental processing of the information received through hearing, and this could impair even further the communication skills of these patients.The goal of this paper is to check the Auditory Processing of temporal lobe epilepsy patients, as to the mechanisms of: discrimination of sounds in sequence and of tonal patterns, discrimination of the sound source direction, and recognition of verbal and non-verbal sounds in dichotic hearing. Thus, we try to understand the effect of epileptic spells in the auditory processing of individuals with this neuronal disorder, aiming at improving their language rehabilitation process.This study was carried out in the Department of Hearing Disorders of the Federal University of São Paulo - Paulista School of Medicine (UNIFESP-EPM), after being approved by the Research Ethics Committee of the same University (515/00). The assessment started after the individuals read the Information Letter about the study goals and signed the Informed Consent Form to participate in the study. This was designed to be a “case-controlled study”.35 patients were referred from the Department of Epilepsy of the Neurology Center of the São Paulo University Hospital. Of these, only 13 came to our department for Auditory Processing Assessment. Notwithstanding, five patients were withdrawn from the sample because they had epilepsy spells that were not restricted to the temporal lobe or did not have a confirmed diagnosis of temporal lobe epilepsy. We then assessed eight male and female patients, with ages varying between 22 and 51 years (Group I). All the individuals who took part in the study were diagnosed with Temporal Lobe Epilepsy and had their diagnosis confirmed by the Department of Neurology of the University . They alThe patients underwent a medical interview and later a basic audiologic evaluation, comprising threshold tonal audiometry, speech recognition threshold (SRT), acoustic immitance measures and acoustic reflex thresholds. This assessment was carried out in order to check on the existence of any peripheral alteration that could interfere in the patient's performance in carrying out central behavioral tests. None of the patients showed any alteration that would exclude him/her from the study.1)Sound Location Test: according to Pereira (1993)2)Recognition Test of the Duration Pattern: according to Musiek3)Digits dichotic test - which assesses the identification behavior, through verbal repetition, familiar words presented at a dichotic hearing, in other words, at the same time, a different one for each ear. We used the recorded version according to what Pereira described (1997)4)Non-Verbal Dichotic Test: assesses the identification behavior, by pointing to representative figures, one between two non-verbal sounds presented at the dichotic hearing, in other words, simultaneously, a different one for each ear. We used the recorded version, according to Pereira (1997)The individuals were then submitted to four behavioral tests in order to assess their auditory processing:At the Free Attention Stage, the patient would freely choose which of the two sounds he/she would identify among the 24 pairs of stimuli presented. At the Right Side Guided Hearing Stage (EDD) the patient should identify only the sound stimuli hear through the right ear and at the Left Side Guided Hearing Stage (EDE) he/she should only identify the sound stimuli heard through the left ear. At the guided hearing stages, 12 pairs of stimuli were presented. As a reference for normality, we accepted the symmetry of responses, according to Ortiz, Pereira, Vilanova (2003)The procedures used to carry out the tests, as well as the reference values for normality followed those used in the Hearing Processing Outpatient Ward at the Department of Hearing and Speech Sciences of the Federal University of São Paulo.In order to assess how the mental processing of information received through hearing occurs, we used a set of tests encompassing the different auditory mechanisms described by Guyton & Hall, 1997The results attained were compared to those from a group of individuals without neurological impairments, called Group II. Thus, four males and six females, with ages matching those individuals from Group I , underweIn order to include Group II individuals, the following criteria were used: no complaints and/or symptoms related to epilepsy or other neurological alteration and presence of responses to threshold tonal audiometry and acoustic immitance within normal limits.In order to analyze the results, we chose a descriptive statistics approach, calculating the average values of right answers, in percentages, even because the small sample size of individuals with temporal lobe epilepsy made it unfeasible the use of conventional statistical tests.Individuals with temporal lobe epilepsy whom comprised the Group I had their performances similar to those individuals from normo-hearing comparison group, whom comprised Group II, as to the sound source discrimination mechanism .As we analyzed the results from the Sound Location Test, selected to assess the Sound Source Direction Discrimination mechanism and the sound source location capability, we noticed that  100% of In the specialized literature we see that the structures responsible for sound location are the superior olivary complex and the inferior colliculus, located at the Brain StemBellis (1996) described sound location as a function of binaural interaction, and it reflected the way through which the information coming from each ear interact, in other words, how they are processed together. Alterations in this capability would be justified by an asymmetrical peripheral hearing loss or alterations in the hearing processing that takes place in the Brain StemThe literature also has reports of complex sounds being used in sound location tests, such as the one used in the present research study. This type of sound would be more easily located than pure sounds in carrying out this type of testThe findings of our study are in agreement with those from Shankweiler (1961), who did not see performance differences in those individuals with alterations in the temporal lobe when compared to normal individuals or individuals with alterations in other brain regionsIndividuals with temporal lobe epilepsy, Group I, presented lower performance than the comparison group of normal-hearing individuals who comprised Group II  as to thIn this paper, the performance from Group I and II individuals in the Duration Pattern Test  and 5 waThus, we observed that individuals from Group I presented lower performances when compared to those from Group II as discrimination mechanisms for sounds in sequence and tonal patters  are concerned. Most individuals from Group II had impairments in this mechanism.We know that through the Duration Pattern Test it is not possible to determine the side of the lesion/dysfunction, since research has shown that results were altered in both ears in individuals with brain lesions, regardless of the side affectedIn the present study, it also became clear that regardless of the epileptic spell side, the individuals had a deficit in the performance of tasks in this test, in both ears, without differences between them; similar to what has been found in previous studiesAs we analyze the performance of individuals from Groups I and II in assessing the selective attention hearing mechanism for verbal sounds using the Digits Dichotic Test  and 7, wThe studies found reported a preference of the Left Hemisphere (HE) for the processing of verbal sounds, thus, a certain advantage of the right ear (OD) was observed in this type of task for speech stimuli, using syllables, words, logatomas, phrases or digitsBroadbent (1954), when introducing the study with a dichotic task, also observed that when they reproduced the stimuli presented to both ears, individuals would tend to first answer all the stimulus of one ear and, afterwards, of the other earKimura  stated that the contralateral auditory pathways are more efficient than their ipsilateral counterparts and, therefore, for a dichotic task the stimuli is more easily processed by the contralateral ear to the prevailing brain hemisphere for speech sound processingPrevious studies have shown that the dichotic stimulation in individuals with epilepsy makes the message processing depend on factors such as the presence or absence of brain lesion and the side in which they have the spell or the lesion, in other words, in the presence of cortical lesion, the preference of information processing would be in the contralateral side to it (effect of the lesion); in its absence, it would occur in the hemisphere ipsilateral to the neuronal discharge The results attained in the present research agree with those from Dibi 1996) and Ortiz, Pereira, Vilanova 2002) when they observed worse results in the group of individuals with epilepsy when compared to normal subjects in the verbal dichotic task test96 and Or02 when tAnother factor that may have influenced the response of the individuals was the speed at which the stimuli were presented, since the lower the stimuli presentation speed in the dichotic tasks, the less response asymmetry is seenWe also did not observe the presence of a lesion effect, having seen that the only individual from Group I who presented response asymmetry showed a response pattern very different from what is mentioned in the literatureThe Non-Verbal Dichotic Test allows us to assess the selective attention mechanism in a binaural separation task. In this paper we noticed a difference between groups I and II individuals in the performance of this test in the Free Attention Stage. Although response variability was similar among them, more individuals from Group I presented response variabilities  when compared to Group II, in which only 40% of the individuals presented such result  and 9. NIn the Left and Right Guided Hearing Stages, there was no difference in the performance of the groups, in general. In none of the groups we observed a predominance of stimuli recognition presented to one of the ears. However, one individual from Group I (number 4), presented discrepant results in these stages of the Test, moving away from the responses of the remaining subjects  and 11. Data from the literature show that a number of studies carried out with dichotic hearing for non-verbal sounds used tonal, musical and/or melodic stimuliStudies carried out with normal individuals show a clear preference for the Left Ear for the processing of non-linguistic soundsThe OE advantage for non-linguistic sound processing indicates a preference for the right hemisphere (HD)Kimura  reported that the contralateral pathways are more efficient than their ipsilateral counterparts and, therefore, the dichotic stimulation responses are asymmetrical, with advantages for the ear contralateral to the predominant hemisphere in auditory stimulus processing. Moreover, he also described that the very easy stimuli would not cause this asymmetry in their processingAuthors who studied patients with epilepsy saw that patients with proven cerebral lesion presented the “lesion effect”, in other words, a predominance of the contralateral hemisphere to the lesion in sound processing, regardless of it being verbal or not. In the patients that did not show any lesions in their CT-Scan, the specialized literature reported 26 a predominance of stimuli processing by the hemisphere ipsilateral to the spells side. Other studiesThe studied carried out by Ortiz, Pereira, Vilanova (2002) compared the performance of individuals with partial epilepsy spells to those with generalized episodes in regards of the dichotic task with non-verbal sounds. They saw that both Groups presented similar performances, which were worse than that of normal individuals. This study also reported the lack of ear advantage in the nonverbal stimuliThe results attained in this paper corroborate those from Ortiz, Pereira, Vilanova (2003) when they analyzed the Free Attention Stage of Group II individuals, since the author also observed response symmetry in normal individuals in this Test stageThe findings of this paper are also in agreement with those from Spellacy (1969) apud Spellacy, Blumstein (1970) which did not see differences between the ears when they applied a test using environmental stimuliNotwithstanding, these findings are in disagreement from others that did report left ear advantages towards the processing of non-verbal stimuli, such as tonal sequences, melodies, prosodic characteristics and sound effectsAs we analyze the presence of a lesion effect or the paradoxical effect, we noticed that despite the average symmetry of responses between the ears in the Free Attention Stage in both groups, in Group I individuals who presented response asymmetry there was an agreement with the findings reported in the literature, that is, the individual who presented a lesion in the cortical hemisphere presented the “lesion effect”, while the other individuals with response asymmetry presented the “paradoxical effect”In the present study we have noticed that the temporal lobe was a factor that differentiated sound processing and the individuals with this disorder presented a loss in the analysis of their verbal and non-verbal sounds received through hearing, when compared to subjects without cortical alterations. The presence of alterations, either structural or functional in the temporal region caused impairment for these individuals, since this cortical region is responsible for the processing of acoustic information. However, the reduced sample of this study made impossible some analyzes types, such as the use of medication (mono or polytherapy), frequence of spells, age of onset and type of crisis, besides the use of statistical tests to compare the responses when of the assessment o auditory processing between the Groups. Later studies with a larger number of patients that would allow us to control these variables would broaden the understanding of how the mental processing of auditory information happens in individuals with temporal lobe cortical alterations.The results of this research, which assessed the Hearing Processing in individuals with temporal lobe epilepsy, show that the affected individuals had a similar performance to those age matching controls without cortical damage, as to their sound source direction discrimination hearing mechanism (sound source location), and greater loss in the processing of hearing received information for sounds in sequence and tonal patterns  discrimination mechanism and the recognition of familiar verbal sounds and non-verbal sounds in dichotic hearing (selective attention).


The pathogenesis of diabetic retinopathy in humans remains imperfectly understood; in particular, the kinetics of the various pathogenic events in the very early stages of retinal damage are difficult to recognize. Animal models are useful but they do not recapitulate all aspects of human pathology and in particular are not completely transposable to study macular pathology since the majority of laboratory animals do not have a macula.While for years, diabetic retinopathy has been considered primarily as a microangiopathy, it is becoming increasingly clear that the neurovascular complex and its regulation are affected early in the retina during diabetes and may precede the signs of microangiopathy that are still the first clinically detected signs by simple fundus photography.However, this microangiopathy would already be a fairly advanced stage in the sequence of biochemical and neuronal deregulations secondary to chronic hyperglycaemia in the retina. The neurovascular complex, which includes not only the vessels and interneurons but also the various glial components of the retina, could be the first site of damage. However, to date, there is no clinically measurable parameter that indicates damage to this neurovascular complex in a meaningful way. The recognized criterion in clinical trials is a gain of three lines of visual acuity, which is not suitable for the evaluation of treatments that would aim to protect the retina from neuronal damage before irreversible complications or macular oedema occur. Neuroprotection is necessary to allow patients to preserve vision in the long term independently of the treatments currently active on macular oedema.Professor Simo’s review  nicely eSeveral molecules, known for use in other indications, have effects on retinal function in different models of diabetic retinopathy. This is the case of Tauroursodeoxycholic acid (TUDCA), a bile acid, which showed powerful neuroprotective properties in various models of retinopathy  and in hThe group of Jeff Boatright showed that TUDCA starting 1 week after induction of streptozotocin in mice significantly ameliorated retina function evaluated by electroretinography and optokinetic testing . StartinGlibenclamide, well known for its hypoglycaemic properties, has independent neuroprotective properties in the brain and in the retina ,6,7 as dIn a more novel way in this indication, a depot formulation of Rho kinase inhibitor is proving interesting to protect the vessels but also the pigment epithelium junctions, with an effect on the extent of ischaemia , which fDr Lebon, together with Boehringer-Ingelheim’s group, has assessed the slow release of an inhibitor, previously used in humans to prevent stroke complications, and they showed promising results for the clinical use of this compound . Indeed,Clinically, anti-VEGF treatments have demonstrated their efficacy in reducing oedema and improving visual function at the cost of repeated injections, as highlighted by Professor Lowenstein in her meaningful review . IntraviThe other class of drugs used clinically for diabetic macular edema is corticosteroid therapy, the effects of which would be better if dexamethasone implants were used earlier and even as a first-line treatment. However, the risk of intraocular pressure raise and of cataracts still limit their use in young phakic patients.In the NAVEDEX study, which was a multicenter retrospective observational study, it was demonstrated that good visual gain can still be achieved in patients with low visual receiving the dexamethasone intravitreous implants as first-line treatment .Professor Kodjikian emphasized that Fluocinolone implants have the advantage of lasting several years and are a relevant alternative for diabetics whose retinopathy has been progressing for several years, although the rate of ocular complications is high .In order for new treatments, evaluated in animal models, to find a place in the therapeutic arsenal, it is essential to continue the functional phenotyping of patients at different stages of their disease as recalled in the review of Professor Simo . Recent We thank the authors for the very good reviews and the original work. We also thank the reviewers and the editorial team of Pharmaceutics for their professionalism and efficiency.


Based on the Environmental Kuznets Curve (EKC) hypothesis, this paper examines whether rural broadband adoption affects agricultural carbon reduction efficiency (ACRE), using panel data from 30 Chinese provinces from 2011 to 2019. This paper achieves a measurement of ACRE by taking the carbon sink of agricultural as one of the desired outputs and using a Slacks-Based Measure (SBM) model and the global Malmquist–Luenberger (GML) index. The results show that: (1) Rural broadband adoption has a positive effect on ACRE. The relationship between the income of rural residents and ACRE was an inverted U-shaped, which confirms the EKC hypothesis. (2) Land transfer has a significant promoting effect on the relationship between rural broadband adoption and ACRE. When the land transfer rate is high, the positive effect of broadband adoption is obvious. (3) The positive effect of broadband adoption on ACRE was more obvious when farmers invested more in production equipment, that is to say, it has a significant positive moderating effect. As farmers in many developing countries suffer from increasingly frequent and severe extreme weather events, we believe that the results of this study also have implications for the implementation of agricultural carbon reduction and smart agricultural equipment roll-out in many countries. In the past, due to a large population and limited arable land, China’s agricultural industry focused strongly on extensive farming. China’s high-input, high-consumption, and high-pollution agricultural development model has resulted in significant pressure on resources and the environment, threatening the sustainability of agriculture [Communication Industry Statistical Bulletin, by the end of December 2019, the net increase in rural broadband users was 17.36 million, which was 14.8% higher than the previous year. This growth rate was 6.3 percentage points higher compared to urban broadband users in the same year. Furthermore, digital technology is spreading rapidly into agriculture. Agricultural informatization has become an important power source to promote the high-quality development of China’s agriculture. Therefore, embedding ICTs as external technology and studying their impact on agricultural carbon reduction efficiency (ACRE) is key to testing the actual effect of the “Digital Villages” project in China.The sustainable development of agricultural systems has become a more urgent issue in the context of global challenges such as the COVID-19 pandemic and food insecurity. Climate change has substantial impacts on water balance, affecting the surrounding industries, agriculture, and other economic sectors . Carbon e effect . Therefoiculture . In ordeiculture ,6. The piculture ,8. Digiticulture . As a de2 emissions generated by manufacturing or other human activities and the optimal CO2 emissions [2 emissions of agricultural production activities to the actual CO2 emissions under fixed input-output conditions. ACRE directly reflects the effect of regional agricultural CO2 emissions and indirectly reflects the potential of regional agricultural CO2 emissions. The literature on ACRE measurement is mainly divided into three parts: One is the selection of the measurement model, another is the selection of measurement index, and the final one is the selection of measurement indicators. First, for model selection, the Solow residual, Algebraic Index, and Stochastic Frontier Approach (SFA) can only fit the production process of one kind of output. In contrast, the Data Envelopment Analysis (DEA) method does not need to set a specific form of production function, but the traditional DEA model has one distinct limitation: non-effective units can only rely on radial improvement to reach the frontier; that is, increasing or decreasing the input and output in the same proportion. In order to overcome this limitation of the traditional DEA model, Tone [2 emissions [Three core issues are being discussed in existing literature on agricultural carbon reduction and its relationship with ICTs. The first core issue is of updating the measurement index and method for ACRE. Carbon reduction efficiency (CRE) is a popular concept in industrial and urban economies and is often used to measure the gap between the actual COmissions ,12. Accoel, Tone  proposedel, Tone ; and theel, Tone  which inel, Tone  showed tmissions ,18,19.2 emissions and changes in food demand. First, regarding CO2 emissions, micro measures to combat this issue focus on changes in land use type and the return of grassland and farmland to forest [2 emissions reduction at the macro level [The second core issue is the identification of the main factors driving agricultural carbon reduction. At present, the literature mainly focuses on the sources of agricultural COo forest , as wello forest ,22. Agriro level . It was ro level  found Kuro level ,26. Empiro level ,28, thusThe third core issue is the relationship between ICTs and agricultural carbon reduction (sustainable agriculture). In practice and theory, it is indisputable that ICTs directly affect the productivity of agriculture ,30. In tThe above three core issues for research and progress have important theoretical value and policy implications for analyzing the impact of ICTs on ACRE. Indeed, broadband is the main infrastructure for carrying information, and household broadband penetration within a region is often used to represent ICTs development level ,37,38. OThis paper argues that there is currently a lack of discussion on the mechanism by which rural broadband adoption affects agricultural carbon reduction, which may dilute its importance in agricultural sustainability. Therefore, in the context of China’s “Digital Villages” project and carbon reduction strategy, this paper uses provincial panel data and econometric modeling from 2011 to 2019 to verify the impact and moderating effect of ICT application on ACRE. The results show that in accordance with the environmental Kuznets Curve (EKC) hypothesis, rural broadband adoption has a positive effect on ACRE. In addition, we also find that higher rates of broadband access are associated with greater improvements in ACRE through increased land transfer rates. With the improvement of informatization, farmers’ investment in production equipment also plays a positive role in ACRE.Compared with previous studies, the main contributions of this paper are as follows. (1) It confirms the positive impact of rural broadband adoption on ACRE, complementing existing evidence on the influencing factors of ACRE. (2) This paper analyzes the moderating effect of land transfer and farmers’ investment in production equipment on ACRE in the context of rural broadband adoption, and the conclusion has a certain reference value for the promotion of land transfer and smart agricultural equipment decision-making. (3) Our paper takes the carbon sink of agricultural production as one of the desired outputs and uses SBM modeling and the GML index method to measure ACRE more comprehensively.This paper is organized as follows. After the Introduction, 2 emission reduction is divided into two strands. The first strand is direct effects. The ultimate goal of new technologies is reflected in the long-term sustainability of agriculture, and broadband adoption provide a foundation for precision agriculture. The use of precision-agriculture-applied automated management technology systems and geographic information systems has led to increased yields, while also reducing the adverse impact on the environment [This study analyzes the EKC hypothesis considering the role of broadband adoption. The nexuses between rural broadband adoption and agricultural carbon reduction are complex. The micro-mechanism of broadband adoption influencing agricultural COironment . Environironment ,43, and ironment . Unfortuironment . In previronment  and Ma aironment  found a The second strand is the propagation effect. Residents trust public positive information the most and trust private negative information the least . The sprHypotheses 1 (H1).Rural broadband adoption directly promotes the improvement of ACRE.2 emissions. Based on the estimation of agricultural CO2 emissions in China from 1991 to 2018, the EKC model is used to conclude that economic and income growth is the main driving factor of agricultural CO2 emissions [The EKC hypothesis illustrates that rising income contributes to pollution but after up to a point, after which pollution decreases. However, pollution changes with income due to scale, composition, and technique effects ,51. Somemissions . In summHypotheses 2 (H2).The income of rural residents has a non-linear effect on ACRE.2 emissionsTheoretically, the mismatch of production materials directly inhibits output and distorts the input decisions of micro subjects, resulting in the loss of environmental welfare . Under tThe widespread development of broadband adaption around 2010 enabled precision agriculture to develop web services resulting in information equipment such as spraying drones and soil temperature sensors . SeveralHypotheses 3 (H3).Land transfer plays a positive moderating role between broadband adoption and ACRE.Hypotheses 4 (H4).The investment of farmers in production equipment plays a positive moderating role between broadband adoption and ACRE.Based on the above research hypothesis, the theoretical analysis framework of this study is obtained .Broadband). Finally, the control variables and moderator variables include rural disposable income (Income), rural disposable income squared (Income square), operation scale , agricultural economic status (Status), industrial added value , the ratio of disaster (Damage), land transfer (Ltr), and equipment investment (Equipment). The above data mainly come from the China Rural Statistical Yearbook, China Statistical Yearbook, China Agricultural Machinery Industry statistical Yearbook, and China Environmental Statistical Yearbook.In order to investigate the direct impact of rural broadband adoption on ACRE and its related moderating effect, this paper uses panel data of 30 provinces  from 2011 to 2019. First, the dependent variable is ACRE, which is measured using input and output data. Second, the core independent variable is the rural broadband adoption ratio  index method to calculate ACRE [2 sink of agricultural production are taken as desirable outputs, and the undesirable output is CO2 emissions generated in the process of agricultural planting.By definition, ACRE should be measured according to the actual level of agricultural carbon emissions and the theoretical optimal COate ACRE ,59,60,61ate ACRE , the totThe reasons for choosing this approach are as follows. First, the GML index method can simulate multiple inputs and outputs simultaneously to accurately measure ACRE. Second, the SBM model can measure efficiency from multiple angles, evaluate the impact of non-zero input and non-zero output relaxation, and comprehensively measure ACRE. Finally, in view of the above advantages, the SBM model and GML index method are suitable for estimating the efficiency of agricultural carbon emissions reduction.Referring to Meng and Qu , accordi2 emissions reduction , based on the calculation of the SBM model, the GML index is determined as follows:In Equation (1), 2 reduction efficiency in each province. 2 emissions and the minimum possible CO2 emissions, and therefore implies greater redundancy in agricultural carbon emissions and lower efficiency of carbon emissions reduction.In Equation (2), Broadband. In the robustness test, the penetration rate of household computers in rural households, i.e., the ownership of household computers per 100 households, is selected for reference Zhang [Computer. This variable is selected because rural households need Internet terminal equipment to install fixed broadband.In the Introduction, the impact of ICT application on agricultural carbon reduction was described in detail. Broadband penetration, as a measure of ICT development, is usually measured by the proportion of broadband connections available ,63 Therece Zhang  and is r2 emissions. The EKC model estimates that economic growth is the main driving factor of agricultural CO2 emissions in China from 1991 to 2018 [2 sources  showed an obvious U-shaped trend with increasing farmland size [Scale. Industrial agglomeration is closely related to economic development [Status. With increasing industrial agglomeration and industrialization, rural production and living equipment will be effectively improved, providing material conditions and product markets for agriculture and promoting high-quality development of the agricultural economy. Therefore, in this paper, the industrial added value of each region is used to measure the degree of industrialization of the region, with the variable being Industrialization. In addition, rural economic development is often affected by natural disasters [Damage. Finally, moderating variables are chosen. Theoretically, mismatches in production materials directly inhibit output and distort the input decisions of micro subjects, resulting in loss of environmental welfare [Ltr and Equipment, respectively. The factors affecting ACRE are complex, so we also added control variables. Some previous studies have focused on the non-linear effects of the economy and income on agricultural CO to 2018 . Hence, and size . Therefoelopment ,67,68. Tisasters . Agriculisasters , thus af welfare . TherefoThis paper constructed the following econometric model to analyze the impact of broadband adoption on agricultural carbon emissions reduction efficiency by referring to Tang et al. .(3)ACRE2 reduction efficiency in each province per year, and i as a proportion of time t. In Equation (3), Ltr) and equipment investment (Equipment), based on the above benchmark model. The specific model is constructed as follows:In order to analyze the moderating effect, this paper adds two moderating variables, land transfer .Equations (4) and (5) are the measurement models of Communication Industry Statistical Bulletin, the proportion of fixed broadband access users in 2019 was 30%, an increase of 1.2 percentage points from the end of the previous year. Therefore, there remains much room for improvement in rural broadband access. The minimum number of computers per 100 households in rural areas was about 4 and the maximum number was about 75. This indicates a certain regional gap in the terminal application of rural broadband.Descriptive statistical results of variables are shown in In Broadband) was 0.201 and significant at 5%. Income had a significant negative impact on ACRE, that is, within a certain range of income, the income of rural residents significantly reduces the ACRE. However, Income squared had a significant positive impact on ACRE, such that when income exceeded a certain range, the increase in the income of rural residents was conducive to improving ACRE. This confirms the U-shaped relationship between rural residents’ income and ACRE, consistent with the EKC hypothesis. At the same time, this result preliminarily verifies H1 and H2. Broadband adoption significantly promotes ACRE. Models (2) and (3) report the results after adding other control variables. It can be seen that Broadband had the same positive impact on agricultural carbon emission reduction efficiency as the previous model, and it is significant at 5%. This indicates that the results in H1. This may be because broadband adoption improve ACRE by promoting technological innovation, alleviating the distortion of industrial structure, and improving the efficiency of resource allocation [2 emissions [The purpose of this study was to investigate the impact of broadband adoption on agricultural carbon reduction efficiency. location . In thislocation . Second,missions . Therefomissions .In addition, the scale of operation significantly affected ACRE. Possible reasons are as follows. First, the transfer of surplus agricultural labor concentrates cultivated land in the hands of the operators, causing a scale effect. This improves agricultural output, which is conducive to improving the enthusiasm of farmers for production standardization. Second, it provides intensive space for the large-scale socialization of agricultural services. Fragmented farmland is not conducive to increasing the scale of services, while larger scale production promotes cost reductions for green agricultural services. Agricultural economic status had a significant negative effect on ACRE. The reasons may be as follows. Areas with high agricultural economic status are the main grain-producing areas. With the goal of ensuring grain output, the concentrated input leads to pollution. Compared with non-major grain-producing areas, agricultural production activities in major grain-producing areas are more concentrated, and agricultural non-point source pollution per unit space increases with increasing production scale, resulting in greater environmental pollution. The degree of industrialization also had a significant effect on ACRE. This may be due to the crowding out of agriculture by industry to some extent. The development of industry requires the expansion of land in a large area, causing huge resource pressure for the development of agriculture. The frequency of natural disasters also negatively impacted ACRE. This may be explained by direct effects on the expected output and an impaired ability of agricultural activity to sequester carbon. Natural disasters may also negatively affect the motivation of farmers, including for green agricultural production. Therefore, considering agricultural development and ecological environmental protection under the goal of food security is a mainstream pursuit for current agricultural development.In order to verify the accuracy of the conclusion, it is necessary to test the robustness of the conclusion and replace the main independent variables. The computer penetration rate of rural residents  was used to replace the ratio of broadband adoption. The core of this study is the nexus between broadband adoption and ACRE. However, an underlying endogeneity problem may occur. On one hand, there may be a reverse causality. If ACRE is higher in one province, it will have a spillover effect on the rest of the country, leading to a larger proportion of broadband adoption in other provinces. On the other hand, there may be some missing variables or measurement errors. These problems all lead to the underlying endogeneity problem. In order to alleviate and solve this problem, this paper uses the independent variable of a single lagged period and the heteroscedasticity-based recognition strategy for further estimation.Referring to Yu et al. , this pap-value of the Sargen-Baseman test statistic is greater than 0.05, which shows that all instrumental variables are exogenous and valid. Based on the above tests, it is concluded that the influence coefficient of broadband adoption on ACRE is 0.563, which is significant at the level of 5%. These results again demonstrate that the adoption of broadband can promote ACRE.To solve the endogenous problem, the heteroscedasticity-based recognition strategy from Lewbel  is adopt2 intensity and spatial heterogeneity. Therefore, this paper further analyzes the moderating effects of land transfer (Ltr) and equipment investment (Equipment) on ACRE under the effect of broadband adoption, including further analysis of possible heterogeneity.Through the robustness and endogeneity analyses above, the relationship between rural broadband adoption and agricultural carbon emission reduction efficiency has been verified. However, existing studies have shown that compared with small farmers, land transfer operators are more likely to accept and adopt low-carbon agricultural technologies . InformaBroadband•Ltr) is greater than zero and achieves statistical significance at the 10% level. This means that a higher proportion of broadband adoption results in improved ARCE by increasing land transfer rate. This conclusion verifies H3. A possible explanation for this phenomenon is that land transfer promotes the possibility of scaling up operation, causing a scale effect and improving agricultural output. This is conducive to improving the enthusiasm of farmers for production standardization and also increasing investment in green technology to provide an economic basis for promoting green agricultural production.Broadband•Equipment) has an impact factor greater than zero and passes the significance test at 10%. Therefore, improvements in informatization level support greater farmers’ investment in production equipment, thus increasing ACRE. This conclusion verifies H4. This can be attributed to the increased accessibility to precision agriculture that is provided by broadband adoption. While equipment investment with low-level informatization cannot realize precision agriculture and therefore result in increased CO2 emissions, the adoption of broadband in rural areas facilitates greater precision agriculture, supporting yield growth and the rational use of inputs. Thus, precision agriculture supported by broadband adoption minimizes the adverse impact on the environment.The moderating effect of farmers’ investment is reported in model (9). The interaction  China is vigorously implementing the “Digital Villages” strategy, and a significant quantity of broadband infrastructure investment is gradually meeting the requirements of rural production and life. Therefore, future work will expand the application of ICTs at the production end of rural areas, advocate the use of broadband to connect farmers with green production and management and improve the efficiency of resource utilization. (2) The government should consider environmental externalities when formulating income redistribution policies. Transferring the surplus rural labor force can increase the income of rural families and enhance awareness of rural environmental protection, to realize the “win-win” of environmental protection and economic development. (3) Improving the land transfer rate is beneficial to increase the investment of green technology. At the same time, governments should continue to increase investment in intelligent agricultural production equipment. On the basis of the existing broadband infrastructure in rural areas, we should vigorously promote the use of information agricultural production equipment that is suitable for agricultural production, convenient for farmers, low cost, and simple to operate. (4) Rural broadband construction has lagged behind urban areas for a long time , so extrFinally, it should be acknowledged that this work has some limitations. First, due to the lack of relevant data, our study only used broadband penetration to measure the application of ICTs development. To be more comprehensive, the application of ICTs development is measured in terms of infrastructure construction and utilization of ICTs. Therefore, in the following research, we will add indicators such as “network speed” and “Internet usage duration” to construct a comprehensive measurement of ICTs level. Second, this study investigated the impact of the application of ICTs on ACRE from a macro regional perspective. To further analyze the impact of the application of ICTs on the behavior of enterprises and farmers from a micro perspective, more representative samples should be used in future assessments of micro subjects’ behavior.


This study aimed to evaluate the possibilities of artefact reduction using different anatomical implant positions with the Bonebridge bone-conduction hearing implant 602 for a patient with an acoustic neuroma requiring regular diagnostic magnetic resonance imaging of the tumour position.Three implant positions and magnetic resonance imaging examinations with and without customised sequences for metal artefact suppression were investigated. The diagnostic usefulness was rated by a radiologist , and the relation between the area of artefact and the total head area was calculated .p < 0.0001). The customised slice-encoding metal artifact correction view-angle tilting metal artifact reduction technique sequences significantly decreased the relative artefact area between 5.13 per cent and 25.02 per cent. The smallest mean artefact diameter was found for the superior to middle fossa position with 6.80 ± 1.30 cm .Following the qualitative analysis, the radiologist rated the superior to middle fossa implant placement significantly better for diagnostic purposes, which is in agreement with the calculated artefact ratio (The application of artefact reduction sequencing and special anatomical implant positioning allows regular magnetic resonance imaging in patients with the bone-conduction hearing implant 602 without sacrificing diagnostic imaging quality for tumour diagnosis. Especially for the above-mentioned therapeutic options, regular observation of the status or growth of the tumour is required. Magnetic resonance imaging (MRI) is a technique that allows detection of tumors at much smaller sizes than previously possible using computed tomography (CT) or auditory brainstem responses. This imaging technique visualises internal structures of the body using magnetic and electromagnetic fields, which may cause artefacts or other problems with active implantable devices because of interferences from the internal magnet or metal.Acoustic neuromas are benign tumours that may develop on the hearing and balance nerves near the inner ear which, depending on size, affects the hearing of the patient. The incidence has been estimated to be between 7 and 15 people per million and constitutes about 80 per cent of all tumours found in the cerebellopontine angle.et al. estimated that 50 to 75 per cent of pacemaker wearers would need to have an MRI during their lifetime.4 Accordingly, this means that hearing implant users with an estimated implant lifetime of at least 20 years have an almost 100 per cent chance that they would need an MRI at least once during the implant lifetime period. This number increases dramatically for hearing implant users who, for example, suffer from neurofibromatosis type 2 or other tumour conditions such as vestibular schwannoma or acoustic neuroma. If, because of tumour growth or radiation therapy, hearing is affected beyond the possibility of conventional hearing aid treatment and reconstruction surgery , implantable hearing devices may be applicable. Besides cochlear implants, which are indicated for patients with profound hearing loss or deafness, bone-conduction hearing implants and active middle-ear implants may effectively rehabilitate mild-to-moderate or mild-to-severe hearing losses. Based on the presented patient's hearing loss, bone-conduction devices are further exploited.Unlike conventional radiography and CT scanning, MRI examinations do not expose patients to ionising radiation (Organisation for Economic Co-operation and Development (2021), MRI units (indicator); accessed on 9 February 2021). Therefore, MRI has become an increasingly important and widely used diagnostic tool in clinical routine. Todt TM, PontoTM or ‘direct-drive’ transcutaneous: Bonebridge  or indirectly via the skin (‘skin-drive’ transcutaneous: SophonoTM and Baha® Attract).5 Both device categories contain magnetic and electrically conductive materials inducing difficulties when undergoing MRI measurements or even hindering diagnosis if artefacts occur within the area of interest. An artefact sphere of 15 cm in diameter was reported with Bonebridge (bone-conduction hearing implant 601),6 a distance of 5–10 cm was reported with the SophonoTM implant7 and 11.5 cm was reported from the centre of the Baha Attract implant.8All available bone-conduction hearing implant systems are characterised by excellent sound-transmission properties of the skull bone. Sound is picked up by the externally worn audio-processor microphones and converted into vibratory stimuli that are either applied directly to the bone  were discussed with the hospital implant board which recommended, for both audiological and wearing-comfort reasons, the direct-drive options that were then presented to the patient.The motivation for these experiments was a patient with an acoustic neuroma presenting to the clinic with mild-to-moderate mixed and conductive hearing loss. The hearing loss resulted from an incomplete tumour resection because of the intra-operative association of the tumour with a potential facial nerve injury by the neurosurgeon. The patient showed stable disease without tumour growth for two years, but she suffered considerably from the negative impact on quality of life and showed mild signs of depression following the untreated hearing loss. After two years of unsuccessful conventional hearing aid trials, she insisted on an implantable solution. The possible rehabilitation options for her given indication  with the new generation bone-conduction hearing implant 602, especially when metal artefact reduction sequences were applied14 . The imaging artefacts do not only depend on the type of implant and the scanning parameters but also on the position of the implant in relation to the site of interest. Therefore, it is not only MRI safety for hearing implants that is essential but also the possibility to reduce the implant shadow and artefact region to allow for comprehensive clarification of diagnosis.Following thorough explanations and counselling regarding possible artefacts that may hinder her diagnostic acoustic neuroma management, the patient decided to get the active transcutaneous bone conduction implant . Despite being MRI conditional up to 1.5 Tesla as stated above, the reported artefact size of the precursor model, the bone-conduction hearing implant 601, is approximately 15 cm around the implant side and may also be present on the images on the contralateral side of the head.The aim of this study was to evaluate the possible correlation of customised metal artefact reduction sequences with three different anatomical implant positions on artefact size in a cadaver head implanted with the newest generation of active transcutaneous bone conduction implant.The latest generation of active bone conduction implants, the bone-conduction hearing implant 602 was used. It addresses the most reported disadvantage of its precursor model, the bone-conduction hearing implant 601, which was the size of the implanted bone conduction floating mass transducer. The new generation presents with almost half the size of the previous generation, allowing implantation with a drilling depth of 4.5 mm , which makes pre-surgical planning redundant and allows for more individual positioning options, as investigated in this study. The bone-conduction hearing implant 602 device used was provided by the manufacturer . The bone-conduction hearing implant 602 is MRI conditional at 1.5 Tesla without the need to surgically remove the magnet. The MRI scanner was limited to ‘normal operating mode’ (whole body averaged specific absorption rate of less than 2 W/kg); ‘first level controlled operating mode’ was avoided.One fresh frozen cadaver head was obtained from the Institute of Anatomy, Medical University of Vienna, Austria. The fresh frozen condition enabled surgical preparation under conditions close to the intravital situation. Three different anatomical positions were prepared as indicated in The cadaver head was supine positioned without the need of additional fixation in the MRI scanner according to the standard position in routine clinical practice. All scans were obtained in a commercially available 1.5 Tesla MRI scanner applying different specifications  in the T1- and T2-weighted sequences for the measured positions  and 4. Ip < 0.0001) as well as compared with the two classical approaches of middle fossa (p < 0.0001) and sinodural angle (p = 0.009). The two classical approaches significantly differed from each other (p = 0.0136) but were not significantly different when compared with the no reduction sequences applied. Investigating the diameter of the artefact in the T2-weighted area, the slice-encoding metal artifact correction view-angle tilting metal artifact reduction technique sequence reduced the mean artefact width in the superior to middle fossa position from 7.53 ± 1.02 cm (range: 5.90–9.07 cm) to 6.80 ± 1.30 cm (range: 5.42–9.74 cm), which translates to a reduction of 18.65 per cent in artefact width. The classical sinodural angle position exhibited a mean artefact width of 7.20 ± 1.76 cm (range: 3.89–10.48 cm), and in the classical middle fossa position a mean artefact width of 8.45 ± 1.06 cm (range: 6.51–9.91 cm) was found  in the superior to middle fossa position with slice-encoding metal artifact correction view-angle tilting metal artifact reduction technique sequences was significantly reduced compared with no reduction sequences  is the ‘gold-standard’ for tumour detection, but implant artefacts may hinder regular diagnosis of possible tumour growthCorrelation of three implant positions and MRI customised metal artefact suppression sequences were investigatedThe superior to middle fossa implant position of the Bonebridge bone-conduction hearing implant 602 allows for detailed visualisation of the cerebellopontine angleThe authors also concluded that for their research the middle fossa approach allowed for better visualisation of respective brain structures with both implant versions, but the effect was more prominent with the bone-conduction hearing implant 602 device.p < 0.0001) and compared with the two classical approaches of middle fossa (p < 0.0001) and sinodural angle (p = 0.009). The two classical approaches significantly differed from each other (p = 0.0136) but were not significantly different when compared with the no reduction sequences applied. The results suggested that the experimental position superior to the middle fossa seems to be the most favourable placement to ensure diagnostic imaging quality, particularly on the implanted side.The calculated ratio of full head to artefact size in percentage compared with no reduction sequences showed that applying the customised slice-encoding metal artifact correction view-angle tilting metal artifact reduction technique sequences significantly decreased the relative artefact area in the T1-weighted measurements for all three measured positions  compared with no reduction sequences but was especially prominent in the experimental position superior to middle fossa. Similar but less significant results were seen in the T2-weighted measurement where the relative artefact area in the superior to middle fossa position with slice-encoding metal artifact correction view-angle tilting metal artifact reduction technique sequences was significantly reduced compared with no reduction sequences  when compared with the classical sinodural and classical middle fossa approaches. Imaging of intracranial and supra- and infra-tentorial brain pathologies are clinically more valuable than standard diagnostic MRI without any artefact reduction sequences. The sequence for metal artefact reduction enables 1.5 Tesla MRI in patients with the Bonebridge bone-conduction hearing implant 602 device without sacrificing diagnostic imaging quality, particularly on the implanted side.


Circulation of influenza A virus (IAV), especially within poultry and pigs, continues to threaten public health. A simple and universal detecting method is important for monitoring IAV infection in different species. Recently, nanobodies, which show advantages of easy gene editing and low cost of production, are a promising novel diagnostic tool for the monitoring and control of global IAVs. In the present study, five nanobodies against the nucleoprotein of H9N2 IAV were screened from the immunized Bactrian camel by phage display and modified with horseradish peroxidase (HRP) tags. Out of which, we determined that H9N2-NP-Nb5-HRP can crossreact with different subtypes of IAVs, and this reaction is also blocked by positive sera for antibodies against different IAV subtypes. Epitope mapping showed that the nanobody-HRP fusion recognized a conserved conformational epitope in all subtypes of IAVs. Subsequently, we developed a nanobody-based competitive ELISA (cELISA) for detecting anti-IAV antibodies in different species. The optimized amount of coating antigen and dilutions of the fusion and testing sera were 100 ng/well, 1:4000, and 1:10, respectively. The time for operating the cELISA was approximately 35 min. The cELISA showed high sensitivity, specificity, reproducibility, and stability. In addition, we found that the cELISA and hemagglutination inhibition test showed a consistency of 100% and 87.91% for clinical and challenged chicken sera, respectively. Furthermore, the agreement rates were 90.4% and 85.7% between the cELISA and commercial IEDXX ELISA kit. Collectively, our developed nanobody-HRP fusion–based cELISA is an ideal method for monitoring IAV infection in different species. Influenza is an acute infectious disease caused by the influenza virus (IV), which can rapidly evolve and can cause significant morbidity and mortality , 2. The Many serologic testing assays have been developed for detecting anti-IAV antibodies in the serum samples. These assays include hemagglutination inhibition (HI), agar gel immunodiffusion, virus neutralization test, immunofluorescence assay (IFA), and ELISA , 14. Of There are indirect ELISA (iELISA) and competitive ELISA (cELISA), which use the highly conserved IAV-NP as an antigen to detect antibodies against different subtypes of IAVs in different species . HoweverNanobodies, or named single-domain antibodies, are a novel type of engineered antibodies derived from the variable domain of the heavy chain of heavy-chain antibody (VHH) . NanobodEscherichia coli system. Then, H9N2-NP was used as an antigen to immunize the Bactrian camel and to screen the nanobodies obtained. In total, five nanobodies against H9N2-NP were obtained and modified to contain an HRP tag. Subsequently, the crossreactivity of these nanobodies with other subtypes of IAVs was assessed, and a nanobody recognizing a common epitope located in the NP of all IAV subtype strains was characterized. Then, using this nanobody-HRP fusion as a reagent, a cELISA was developed to qualitatively detect anti-IAV antibodies in the sera from different species. Our study provided a type of cELISA with rapid and easy operation for the diagnosis of IAV infection in different species. Importantly, the method produced was low in cost for following commercial production without the HRP-labeled antibodies in vitro.In the present study, the NP from a subtype of the H9N2 avian IV  was expressed using the A). Western blotting analysis revealed that the recombinant H9N2-NP specifically reacted with positive chicken serum samples for anti-H9N2 AIV antibodies (B), indicating that the recombinant proteins possessed antigenicity.To immunize the camels, anti-H9N2-NP nanobodies were screened and the cELISA was established; the recombinant H9N2-NP was expressed using a bacterial system. SDS-PAGE analysis showed that the recombinant protein was successfully expressed and purified using a nickel–nitrilotriacetic acid (Ni–NTA) column with the expected size of 56 kDa A. Westertibodies B, indicaA). Next, a phage display library containing 9.8 × 108 individual transformants was successfully constructed using the peripheral blood lymphocytes (PBLs) from the immunized camel. Subsequently, the results showed that the positive rate of the library was 98% by colony PCR assay (B), and each clone from the selected 96 clones contained a different VHH sequence (data not shown). The high capacity and positivity rate of the library confirmed the suitable heterogeneity of the library.After the camel was immunized with the H9N2-NP, the titer of antibody against the protein in serum samples reached 1:128,000 based on iELISA A. Next, CR assay B, and eaC). These 94 positive clones were sequenced, and five nanobodies were obtained based on the complementarity-determining region 3 of the sequences. They were named H9N2-NP-Nb5, H9N2-NP-Nb16, H9N2-NP-Nb18, H9N2-NP-Nb42, and H9N2-NP-Nb53. Alignment results suggested that the five nanobodies possessed typical hydrophilic amino acid substitutions in the framework-2 regions (D). In addition, the five nanobodies were tested by iELISA using H9N2-NP and Newcastle disease virus (NDV)-NP separately as coating antigens. The results showed that they specifically reacted with the H9N2-NP (E) but not with NDV-VP, which was expressed with the same procedures and systems as H9N2-NP . Using the supernatant from the transfected HEK-293T cells as testing antibodies, the results of direct ELISA showed that all five nanobody-HRP fusion proteins were secreted and subsequently termed H9N2-NP-Nb5-HRP, H9N2-NP-Nb16-HRP, H9N2-NP-Nb18-HRP, H9N2-NP-Nb42-HRP, and H9N2-NP-Nb53-HRP (B). The titer of the H9N2-NP-Nb5-HRP fusion proteins in the supernatants was 1:10,000, and the titers of the other four H9N2-NP-nanobodies-HRP fusions were 1:1000 .Based on the aforementioned descriptions, the five anti-H9N2 nanobodies fusing with HRP were successfully expressed using human embryonic kidney 293T (HEK-293T) cells as confirmed by IFA identification Fig. 2A. Using E. coli system with the expected size and was successfully purified using a Ni–NTA column (A). The results of direct ELISA showed that the H9N2-Nb5, H9N2-Nb16, H9N2-Nb18, H9N2-Nb42, and H9N2-Nb53-HRP could crossreact with the NPs of subtypes H1N1, H3N2, H5N1, and H7N9 IAVs but not react with the NP of B/Yamagata/16/1988 (B). In addition, each positive serum sample for anti-H9N2, -H1N1, -H3N2, -H5N1, and -H7N9 antibodies confirmed using the commercial IDEXX ELISA kit was detected using these nanobody-HRP fusions as reagents by blocking ELISA. The results showed that the percentage inhibition (PI) of cELISA using H9N2-NP-Nb5-HRP as a probe reached 80% (C). The aforementioned results indicated that the epitope recognized by the H9N2-NP-Nb5 may be conserved across different subtypes of IAVs. Based on these results, H9N2-NP-Nb5-HRP was selected for identification of its recognizing epitope to analyze the conservation in all subtypes of IAVs and for developing the cELISA.To analyze the five nanobody-HRP fusion protein crossreaction with other subtypes of IAV and influenza B virus (IBV), the direct ELISAs using the NP of H1N1, H3N2, B/Yamagata/16/1988, and killed denatured H5N1 and H7N9 particles as the coating antigens were performed. SDS-PAGE analysis showed that the NP of H1N1, H3N2, and B/Yamagata/16/1988 was successfully expressed using the A column A. The re/16/1988 B. In addched 80% C. The afA). After these fragments were expressed using the E. coli system, SDS-PAGE and Western blotting analysis showed that they were successfully expressed with the expected size and purified by a Ni–NTA column (B and C). The results of direct ELISA using these truncated fragments as coating antigens showed that the H9N2-NP-Nb5-HRP reacted with fragments spanning 1 to 1308 bp (N436 amino acids), 1 to 1215 bp (N405 amino acids), 1 to 1182 bp (N394 amino acids) and 1 to 1167 bp (N389 amino acids) but not with 1 to 747 bp (N249 amino acids), 747 to 1494 bp (C249 amino acids), 1 to 1122 (N374 amino acids), 373 to 1494 (C374 amino acids), 187 to 1494 bp (C436 amino acids), or 1 to 1152 bp (N384 amino acids) (D). These results suggested that the epitope recognized by H9N2-NP-Nb5-HRP may be a native conformational epitope and the two regions of amino acids 1 to 62 and amino acids 385 to 389 may be important domains for maintaining the conformation.To identify the epitope recognized by H9N2-NP-Nb5-HRP, different truncated and overlapping H9N2-NP fragments were designed A. After A column , B and Co acids) D. These A). Based on the reaction of different fragments and predicted results, the amino acids located in 388 to 389 (H9N2-NP388–389M), 385 to 389 (H9N2-NP385–389M), and the three regions (H9N2-NP3M) predicted by the docking model were mutated into alanine (A) and expressed using the E. coli system. In addition, the W95, T96, and K100 of H9N2-NP-Nb5-HRP were also all mutated to A (termed H9N2-NP-Nb53M-HRP). SDS-PAGE and Western blotting showed that the mutants of H9N2-NP were successfully expressed with the expected sizes (B and C). Furthermore, the direct ELISA results showed that H9N2-NP3M did not react with H9N2-NP-Nb5-HRP, and H9N2-NP-Nb53M-HRP did not react with H9N2-NP. In addition, although H9N2-NP385–389M still reacted with H9N2-NP-Nb5-HRP, the absorbance at 450 nm value of direct ELISA using the coated antigen was significantly lower than that of H9N2-NP . So, the results of direct ELISA using the mutated proteins indicated that the predicted amino acids for interaction between H9N2-NP and H9N2-NP-Nb5-HRP were correct. Furthermore, these results also suggested that the epitope recognized by the H9N2-NP-Nb5-HRP was a native conformational epitope.To precisely define the epitope, AlphaFold2 server was used to predict the structure of proteins . The docFig. 5E).To further analyze the amino acid conservation of the epitope, the amino acids 354 to 356, 385 to 387, and 445 to 449 of the NP from different IAV strains were aligned. Sequence alignments showed that the epitope was highly conserved among the different subtypes of IAV strains E.The conditions of cELISA using the H9N2-NP-Nb5-HRP fusion as the probe were optimized. Using checkerboard titration experiments with the direct ELISA, the results showed that the optimized amount of coated antigen was 100 ng/well and the optimal dilution of H9N2-NP-Nb5-HRP was 1:4000 . Using tThe average PI (X) value of 180 negative sera from humans, pigs, and chickens detected by the developed cELISA was 1.0%, and the SD was 4.4%. The cutoff value of the cELISA was 14.2% (1.0% + 3 SD), indicating that the PI values of the tested sera by the cELISA were more than 14.2%, suggesting that the serum sample was positive for the anti-IAV antibody. If the PI value was less than 14.2%, this would then suggest they were negative.A). These indicated that the limits of detection of the cELISA for testing sera were 1:320.The results of testing different dilutions of positive sera for anti-IAV antibodies with the cELISA showed that the sera at a dilution of 1:640 was negative, and those at 1:320 were all positive A. These B).The results of testing the positive sera for antichicken, pig, and human viruses’ antibodies showed that the PI values of these sera were all below 14.2%, suggesting that the specificity of cELISA was good B.To analyze the reproducibility of the cELISA, five positive and negative sera were tested to evaluate the intra-assay and interassay variabilities. The intra-assay CV of the PI was analyzed in the range of 3.14 to 7.02% with a median value of 5.08%, whereas the range for the interassay CV was 4.69 to 12.73% with a median value of 8.71% . These dC).The results of direct ELISA using the plates and H9N2-NP-Nb5-HRP stored at 4 °C for 0, 30, 60, 90, 120, 150, and 180 days showed that the absorbance at 450 nm values were ∼1.0, indicating that the plates and the competitive agents H9N2-NP-Nb5-HRP remained active. The results of cELISA showed that the PI of cELISA stabilized at 80%, suggesting that the assay exhibited good stability over the past 180 days C.To evaluate the assay for detecting anti-IAV antibodies in different species, the 2155 sera samples from human (n = 660), swine (n = 660), pet dog (n = 524), goat (n = 195), cattle (n = 28), rabbit (n = 44), cat (n = 44), pet dog (n = 524), duck (n = 98), and wild birds (n = 63) were simultaneously tested by the developed cELISA and the commercial IDEXX ELISA kit. For human sera, the positive rates of the cELISA and the commercial IDEXX ELISA kit were 97.58% (644 of 660) and 97.88% (646 of 660), respectively. For pig serum samples, the positive rates of the cELISA and the commercial IDEXX ELISA kit were 82.42% (544 of 660) and 83.33% (550 of 660), respectively. For pet dogs, the positive rates were 25.19% (132 of 524) and 25.95% (136 of 524), respectively. The positive rates of goat sera were 14.36% (28 of 195) and 14.87% (19 of 195), respectively. For cattle, the positive rates for both kits were 46.43% (13 of 28), and the positive rates of rabbit sera for both kits were 31.82% (14 of 44). For cat sera, the positive rates were 2.27% (1 of 44) and 6.82% (3 of 44), respectively. The positive rates of duck sera were 87.31% (227 of 260) and 88.08% (229 of 260), respectively, and for the wild birds, the rates were 27.73% (33 of 119) and 31.93% (38 of 119), respectively . The afoκ values were >0.4) between the cELISA and either the HI test  or the commercial IEDXX ELISA kit . For the challenged chicken sera, the results showed that the positive rates were 73.63% (67 of 91), 61.54% (56 of 91), and 59.34% (54 of 91), respectively  chickens challenged with H9N2 AIV were tested by the cELISA, HI test, and commercial IDEXX ELISA kit. For the clinical chicken sera, the positive rates of the three assays were 57.30%, 57.30%, and 51.35%, respectively . When thectively . The agrectively .Table 2CA). At 5 days postinfection, one sample was positive using the cELISA, whereas all sera were negative with the HI test and commercial IDEXX ELISA kit (B). These results suggested that the developed cELISA was higher in sensitivity than the HI test and commercial IDEXX ELISA kit for detecting anti-IAV antibodies from challenged chicken sera.In addition, the sensitivities of three approaches were also compared. All sera from 7 days postinfection were positive using cELISA, but only four samples were positive with the HI test and two samples were positive with the commercial IEDXX ELISA kit A. At 5 dLISA kit B. These in vitro labeling of mAbs, resulting in poor batch-to-batch consistency . The developed cELISA was a suitable method for surveillance and monitoring of IAV infection in different species.In conclusion, a common and highly conserved epitope in the NP of different subtype IAVs recognized by the H9N2-NP-Nb5 was identified. Next, a cELISA using H9N2-NP-Nb5-HRP as a probe was developed, and it exhibited good sensitivity, specificity, and high consistency with the commercial IDEXX ELISA kit. Importantly, the advantages of the developed cELISA over commercial kits include the elimination of enzyme-labeled antibodies 2.HEK-293T cells were obtained from the American Type Culture Collection and cultured in Dulbecco’s modified Eagle’s medium  supplemented with 10% fetal bovine serum  at 37 °C in a humidified incubator supplied with 5% COA/chicken/Hebei/LC/2008  was propagated in the allantoic cavity of 9-day-old chicken embryonated eggs. The inactivated and purified H5N1 and H7N9 viral particles were kindly provided by Harbin Veterinary Research Institute, Chinese Academy of Agricultural Sciences.The 180 negative sera from humans (n = 65), pigs (n = 60), and SPF chickens  were used to calculate the PI of the developed cELISA. All these serum samples were confirmed using an IDEXX AIV antibody ELISA kit IDEXX and HI assay. The commercial ELISA kit is the most stable and used in laboratory tests at present , 37. And9) and 185 clinical sera were used for the simultaneous detection of three methods  cells (TransGen Biotech). Then, 0.5 mM IPTG was added for inducing the expression of recombinant H9N2-NP at 25 °C for 12 h. After centrifugation at 8000g for 15 min at 4 °C, the bacteria were collected. After the bacterial cells were sonicated, the recombinant protein was purified using an Ni–NTA column . Expression, purification, and antigenicity of the recombinant protein were analyzed by SDS-PAGE and Western blotting.The H9N2 AIV strain HB08 was used as the template for amplification of the viral gene encoding the NP by RT–PCR. The sequences of the primers used for PCR amplification are listed in A 4-year-old male Bactrian camel was immunized with the purified recombinant H9N2-NP (1 mg/ml) with an equal volume of complete Freund’s adjuvant  subcutaneously , 26. FivE. coli TG1-competent cells. The cells were plated on LB agar plates supplemented with ampicillin and d-glucose at 37 °C. The bacterial monolayer was scraped and added to LB containing 20% glycerin and stored at −80 °C. Then, the positive rate and diversity of the library were determined as described previously . Next, the Ficoll-Paque PLUS Lymphocyte Separation Fluid (Greiner Bio-One) was used to isolate PBLs according to the manufacturer’s protocol. Total RNA was extracted from the PBLs and used as templates for reverse transcription to synthesize the complementary DNA. Next, the VHH genes were amplified using nested PCR as described previously , 40, 41.eviously .via M13K07 (5 × 1011 plaque-forming unit/ml) were added and incubated for 2 h at room temperature. Then, the binding phages were eluted with fresh trimethylamine (100 mM) and neutralized with Tris–HCl buffer . The titers of eluted phages were tested by infecting TG1 cells and then used for the next round of rescue and panning. After three rounds of screening, the ratio of the output of the positive phage to the output of the negative phage was calculated to evaluate the enrichment of phages against H9N2-NP. A total of 96 clones were selected and induced by 1 mM IPTG for expression of anti-H9N2-NP nanobodies. The soluble nanobodies were extracted from the periplasm and tested by iELISA using the H9N2-NP as the coating antigen. The NDV-NP was used as a negative control for the iELISA. The protein was also expressed using the same vector pET-28a described in a previous study .For determining the titers of immunized Bactrian camel and specific detection of the periplasm containing the nanobodies, iELISA was performed based on a previous study . Briefly3. For identification of expression, the transfected cells were analyzed using an indirect IFA with anti-His mAbs as the primary antibody and FITC-labeled goat antimice IgG antibodies as the secondary antibody. Direct ELISA was performed to quantify the secreted expression of nanobody-HRP fusion proteins in the HEK-293T cells as described previously , A/swine/Guangxi/1659/2017 (H3N2), H5N1 (Re-12), H7N9 (H7-Re3) IAV strain, and IBV strain (B/Yamagata/16/1988) were selected for use as coating antigens. Of these, the H1N1 and H3N2 IAVs NPs and IBV NP were also expressed using the bacterial system as for the H9N2 NP and used as coating antigens in the direct ELISA. For H5N1 and H7N9, the purified and denatured viral particles were used as coating antigens.In addition, to further analyze crossreaction, the blocking rate of the nanobody-HRP fusion proteins blocked by the positive sera for antibodies against different subtypes of IAVs to react with the antigens was also determined using the blocking ELISA as described previously . BrieflyA). Based on the nucleotide sequences encoding H9N2-NP, primer pairs were designed for amplifying these fragments  (pymol.org/2/support.html)  . Interacrt.html) .Subsequently, the predicted key amino acids were separately mutated to alanine (A) residues for H9N2-NP and the nanobody to identify the predicted results. The primers used for PCR amplification of the mutated fragments are listed in To analyze the conservation of the epitope among the different subtype IAV strains, the amino acid sequences of the antigenic domains from different strains in GenBank were aligned using the Clustal W module of Lasergene 7.1 . The GenBank accession numbers of different IAV strains were A/chicken/Hebei/0822/2007 (H9N2) (GQ373096.1), A/California/04/2009 (H1N1) (MN371617.1), A/swine/Guangxi/JG1/2014 (H3N2) (MF927881.1), A/HK/212/03 (H5N1) (AY575905.1), A/tree sparrow/Shanghai/01/2013 (H7N9) (KF609528.1), A/swine/Gunma/1/2012 (H1N2) (AB731586.1), A/Berkeley/1/68 (H2N2) (AY210104.1), A/canine/Florida/242/2003 (H3N8) (DQ124158.1), A/mallard/California/57871/2015 (H4N6) (KX351682.1), A/mallard/Sweden/100993/2008 (H7N7) (FJ803195.2), A/swan/Shandong/W4322/2020 (H10N4) (OM373303.1), A/mallard/Beijing/27-MA/2011 (H10N7) (KY688104.1), A/mallard/Korea/SH38-45/2010 (H13N2) (JX030409.1), and A/seagull/Chile/5775/2009 (H13N9) (KF772957.1).Using the selected nanobody-HRP fusion protein as the probe, the cELISA for detecting anti-IAV antibodies in serum samples was designed as described previously . First, Second, the dilutions of testing sera were optimized for the cELISA. The different dilutions  of five positive (P) and negative (N) sera for anti-IAV antibodies  were separately mixed with the optimized dilution of the nanobody-HRP fusion protein. After the optimized amount of H9N2-NP was coated into the plate, the mixtures were added to the plate and incubated for 1 h at 37 °C. Then, the TMB was added, and the absorbance at 450 nm values were read after the reaction was terminated. The optimal dilution of testing chicken sera was determined when the ratio of the positive and negative sera absorbance at 450 nm values (P/N) was the smallest.Third, the incubation times between the mixture, the coated antigen, and the color reaction times after adding TMB were also optimized using the checkerboard assay. The incubation times of 20, 30, 40, 50, and 60 min and the color reaction times of 10 and 15 min were selected. Then, the two optimal times were determined when the P/N value was the smallest.2SO4.After the aforementioned conditions were optimized, the developed cELISA was performed as followed. The 96-well ELISA plates were coated with the optimized amount of H9N2-NP in PBS  at 4 °C overnight. Then, the plates were washed and blocked with 300 μl/well blocking buffer (2.5% milk in PBST) at 37 °C for 1 h. After washing with PBST, 100 μl testing mixture containing the optimal dilutions of serum sample and nanobody-HRP fusion was added to wells and incubated for the optimal time at 37 °C. After washing three times, 100 μl TMB was added and incubated for the optimal time at room temperature, and then the absorbance at 450 nm values were measured after the reaction had been stopped by 3 M HTo calculate the PI of the cELISA, the 180 sera samples negative for anti-IAV antibodies from humans, pigs, and chicken were used. The cutoff value was set to the average PI of 180 negative sera plus three SDs to ensure 99% confidence that the negative sera were within this range.The sensitivity of cELISA was evaluated by testing the different dilutions  of 48 positive sera for anti-H9N2 (n = 16), H5N1 (n = 8), H7N9 (n = 8), H1N1 (n = 8), and H3N2 (n = 8) antibodies.To determine the specificity of cELISA, a total of 181 clinical positive chicken sera against other avian viruses including NDV (n = 44), avian infectious bronchitis virus (n = 27), infectious bursal disease virus (n = 33), avian leukemia virus (n = 23), Marek’s disease virus (n = 21), and avian hepatitis E virus (n = 33), 128 positive pig sera against other swine viruses including porcine reproductive and respiratory syndrome virus (n = 23), porcine epidemic diarrhea virus (n = 27), porcine circovirus (n = 43), and pseudorabies virus (n = 23), and 69 positive human sera against other human viruses including hepatitis B virus (n = 26), severe acute respiratory syndrome coronavirus 2 (n = 15), and human adenovirus (n = 21) were used to test the developed cELISA.The reproducibility of the developed cELISA was analyzed by testing five positive and negative sera to analyze the intra-assay and interassay variabilities. The CV was calculated using the PI values of different sera to evaluate the interassay variation (between plates) and the intra-assay variation (within a plate). Each serum sample was tested using three different plates to determine the interassay CV and three replicates within each plate to calculate the intra-assay CV.The stability of the nanobody-HRP fusion and the developed cELISA were also analyzed. Briefly, the 96-well plates were coated with the purified recombinant H9N2-NP at 4 °C for 12 h. After washing with PBST and blocking with the blocking buffer, the plates were dried in a fume hood and vacuumed. Then, the plates and optimized dilutions of the selected nanobody-HRP fusion were stored at 4 °C. The direct ELISA and cELISA were separately performed as described previously after 0, 30, 60, 90, 120, 150, and 180 days to evaluate the stability.To evaluate the developed cELISA for the detection of anti-IAV antibodies in the sera from different species, the 2155 sera from humans (n = 660), swine (n = 660), goats (n = 195), cattle (n = 28), rabbits (n = 44), cats (n = 44), pet dogs (n = 524), ducks (n = 98), and wild birds (n = 63) were both tested with the developed assay and an IDEXX IAV antibody ELISA kit (IDEXX).Reference methods for detection of anti-IAV antibody in laboratory and literature include HI test, agar gel immunodiffusion, virus neutralization test, IFA, and ELISA , 14. AmoThe 185 clinical chicken sera and 91 sequential sera from 13 SPF chickens challenged with H9N2 AIV stock were simultaneously tested using the developed cELISA, a commercial IDEXX ELISA kit, and the HI test. Then, the coincidence rates were also calculated using Microsoft Excel’s CORREL function.κ index values were calculated to estimate the coincidence between the developed cELISA and the commercial ELISA kit . The LISA kit . These cAll data are provided within the article.The animal experiments were performed in strict accordance with the recommendations in the Guide for the Care and Use of Laboratory Animals of the Northwest A&F University. The protocols used for animal and sera collections were approved by the Institutional Animal Care and Use Committee of Northwest A&F University .This article contains The authors declare that they have no conflicts of interest with the contents of this article.