Dync1li1 is required for the survival of mammalian cochlear hair cells by regulating the transportation of autophagosomes.

Dync1li1, a subunit of cytoplasmic dynein 1, is reported to play important roles in intracellular retrograde transport in many tissues. However, the roles of Dync1li1 in the mammalian cochlea remain uninvestigated. Here we first studied the expression pattern of Dync1li1 in the mouse cochlea and found that Dync1li1 is highly expressed in hair cells (HCs) in both neonatal and adult mice cochlea. Next, we used Dync1li1 knockout (KO) mice to investigate its effects on hearing and found that deletion of Dync1li1 leads to early onset of progressive HC loss via apoptosis and to subsequent hearing loss. Further studies revealed that loss of Dync1li1 destabilizes dynein and alters the normal function of dynein. In addition, Dync1li1 KO results in a thinner Golgi apparatus and the accumulation of LC3+ autophagic vacuoles, which triggers HC apoptosis. We also knocked down Dync1li1 in the OC1 cells and found that the number of autophagosomes were significantly increased while the number of autolysosomes were decreased, which suggested that Dync1li1 knockdown leads to impaired transportation of autophagosomes to lysosomes and therefore the accumulation of autophagosomes results in HC apoptosis. Our findings demonstrate that Dync1li1 plays important roles in HC survival through the regulation of autophagosome transportation.

Thyroid hormone controls the timing of cochlear ribbon synapse maturation.

Ribbon synapses in the cochlear hair cells are subject to extensive pruning and maturation processes before hearing onset. Previous studies have highlighted the pivotal role of thyroid hormone (TH) in this developmental process, yet the detailed mechanisms are largely unknown. In this study, we found that the thyroid hormone receptor α (Thrα) is expressed in both sensory epithelium and spiral ganglion neurons in mice. Hypothyroidism, induced by Pax8 gene knockout, significantly delays the synaptic pruning during postnatal development in mice. Detailed spatiotemporal analysis of ribbon synapse distribution reveals that synaptic maturation involves not only ribbon pruning but also their migration, both of which are notably delayed in the cochlea of Pax8 knockout mice. Intriguingly, postnatal hyperthyroidism, induced by intraperitoneal injections of liothyronine sodium (T3), accelerates the pruning of ribbon synapses to the mature state without affecting the auditory functions. Our findings suggest that thyroid hormone does not play a deterministic role but rather controls the timing of cochlear ribbon synapse maturation.

Reduced Intra- and Inter-Network Functional Connectivity Identified in Patients with Tinnitus with and without Hearing Loss.

INTRODUCTION: The aim of the study was to investigate differences in the intra- and inter-network functional connectivity (FC) of the brain using resting-state functional magnetic resonance imaging (rs-fMRI) in patients with tinnitus, with (T + H) or without hearing loss (T). METHODS: We performed rs-fMRI on 82 participants (21 T, 32 T + H, and 29 healthy controls). An independent component analysis (ICA) was performed to obtain the resting-state networks (RSNs) and calculate the differences in FC. Moreover, we investigated the relationships between networks using functional network connectivity analysis. RESULTS: We identified nine major RSNs, including the auditory network; default mode network; executive control network (ECN), including the right frontoparietal network and left frontoparietal network (LFPN); somatomotor network (SMN); dorsal attention network; ventral attention network; salience network (SN); and visual network (VN). These RSNs were extracted in all groups using ICA. Compared with that in the control group, we observed reduced FC between the LFPN and VN in the T group and between the LFPN and SN in the T + H group. The inter-network connectivity analysis revealed decreased network interactions in the SMN (IC 22)-ECN (IC 2), SMN (IC 22)-VN (IC 8), and VN (IC 14)-SN (IC 3) connections in the T + H group, compared with the healthy control group. Furthermore, we observed significantly decreased network interactions in the SMN (IC 22)-VN (IC 8) in the T group. CONCLUSIONS: Our results indicated abnormalities within the brain networks of the T and T + H groups, including the SMN, ECN, and VN, compared with the control group. Furthermore, both T and T + H groups demonstrated reduced FC between the LFPN, VN, and SMN. There were no significant differences between the T and the T + H groups. Furthermore, we observed reduced FC between the right olfactory cortex and the orbital part of the right middle frontal gyrus, right precentral gyrus, left dorsolateral superior frontal gyrus, and right triangular part of the inferior frontal gyrus within the T and T + H groups. Thus, disruptions in brain regions responsible for attention, stimulus monitoring, and auditory orientation contribute to tinnitus generation.

Discrepancies in Mate Preferences among Chinese Families between Fathers, Mothers, and Children: A Latent Profile Analysis.

Parents develop their own preferences regarding prospective in-laws, which influence their children's mate selection processes and outcomes. From an evolutionary perspective, fathers, mothers, and their offspring have partially divergent genetic interests, potentially leading to conflicts in mate preferences. Currently, the characteristics of discrepancy profiles in mate preferences within Chinese families and their influencing factors remain unclear. Adopting an individual-centered perspective, this study examined the profiles of discrepancies in mate preferences between fathers, mothers, and children across a diverse set of Chinese families, along with their associations with family relations and evaluations of children. This study recruited 337 complete families. The results revealed three distinct profiles of father-mother mate preference discrepancies in families with sons and four profiles in families with daughters. Additionally, both families with sons and daughters displayed three profiles of discrepancies in parent-child mate preferences. Parental perceptions of marital relationships and their evaluations of children were linked to diverse father-mother discrepancy profiles in both families with sons and daughters. The father-son relationship was associated with the profiles of parent-child discrepancies in families with sons, while maternal evaluations of children and daughters' self-evaluations were related to the profiles of parent-child discrepancies in families with daughters. This study provides insights into understanding the conflict patterns and underlying reasons regarding mate preference between Chinese parents and their children within family settings.

Hippo/YAP signaling pathway protects against neomycin-induced hair cell damage in the mouse cochlea.

The Hippo/Yes-associated protein (YAP) signaling pathway has been shown to be able to maintain organ size and homeostasis by regulating cell proliferation, differentiation, and apoptosis. The abuse of aminoglycosides is one of the main causes of sensorineural hearing loss (SSNHL). However, the role of the Hippo/YAP signaling pathway in cochlear hair cell (HC) damage protection in the auditory field is still unclear. In this study, we used the YAP agonist XMU-MP-1 (XMU) and the inhibitor Verteporfin (VP) to regulate the Hippo/YAP signaling pathway in vitro. We showed that YAP overexpression reduced neomycin-induced HC loss, while downregulated YAP expression increased HC vulnerability after neomycin exposure in vitro. We next found that activation of YAP expression inhibited C-Abl-mediated cell apoptosis, which led to reduced HC loss. Many previous studies have reported that the level of reactive oxygen species (ROS) is significantly increased in cochlear HCs after neomycin exposure. In our study, we also found that YAP overexpression significantly decreased ROS accumulation, while downregulation of YAP expression increased ROS accumulation. In summary, our results demonstrate that the Hippo/YAP signaling pathway plays an important role in reducing HC injury and maintaining auditory function after aminoglycoside exposure. YAP overexpression could protect against neomycin-induced HC loss by inhibiting C-Abl-mediated cell apoptosis and decreasing ROS accumulation, suggesting that YAP could be a novel therapeutic target for aminoglycosides-induced sensorineural hearing loss in the clinic.

Aldh inhibitor restores auditory function in a mouse model of human deafness.

Genetic hearing loss is a common health problem with no effective therapy currently available. DFNA15, caused by mutations of the transcription factor POU4F3, is one of the most common forms of autosomal dominant non-syndromic deafness. In this study, we established a novel mouse model of the human DFNA15 deafness, with a Pou4f3 gene mutation (Pou4f3Δ) identical to that found in a familial case of DFNA15. The Pou4f3(Δ/+) mice suffered progressive deafness in a similar manner to the DFNA15 patients. Hair cells in the Pou4f3(Δ/+) cochlea displayed significant stereociliary and mitochondrial pathologies, with apparent loss of outer hair cells. Progression of hearing and outer hair cell loss of the Pou4f3(Δ/+) mice was significantly modified by other genetic and environmental factors. Using Pou4f3(-/+) heterozygous knockout mice, we also showed that DFNA15 is likely caused by haploinsufficiency of the Pou4f3 gene. Importantly, inhibition of retinoic acid signaling by the aldehyde dehydrogenase (Aldh) and retinoic acid receptor inhibitors promoted Pou4f3 expression in the cochlear tissue and suppressed the progression of hearing loss in the mutant mice. These data demonstrate Pou4f3 haploinsufficiency as the main underlying cause of human DFNA15 deafness and highlight the therapeutic potential of Aldh inhibitors for treatment of progressive hearing loss.

Inverse Association of Serum Adipsin with the Remission of Nonalcoholic Fatty-Liver Disease: A 3-Year Community-Based Cohort Study.

PURPOSES: Adipokine alterations contribute to the development and remission of nonalcoholic fatty-liver disease (NAFLD). Adipsin is one of the most abundant adipokines and is almost exclusively produced by adipocytes. However, data on adipsin in human NAFLD are limited and controversial. We performed this study to investigate the association between adipsin and the remission of NAFLD in middle-aged and elderly Chinese adults. METHODS: Whether adipsin is associated with the remission of NAFLD in a 3-year community-based prospective cohort study was investigated. Baseline levels of adipsin were measured in serum samples collected from 908 NAFLD participants. NAFLD was diagnosed using abdominal ultrasonography. Logistic regression analysis and a multiple stepwise logistic regression model including different variables were conducted to evaluate the association between serum adipsin levels and the remission of NAFLD. RESULTS: During a mean follow-up of 3.14 ± 0.36 years, 247 (27.20%) participants with NAFLD at baseline were in remission. At baseline, serum adipsin concentration was positively correlated with body mass index (r: 0.39, p < 0.001), insulin (r: 0.31, p < 0.001), and homeostasis model assessment of insulin resistance (r: 0.31, p < 0.001) and was inversely associated with NAFLD remission with a fully adjusted odds ratio (OR) of 0.28 (0.16-0.48) (p trend < 0.001). In a multiple stepwise logistic regression model, circulating adipsin independently predicted NAFLD remission (OR: 0.284, 95% confidence interval [CI]: 0.172-0.471, p for trend <0.001). The area under the receiver operating characteristic curve was 0.751 (95% CI: 0.717-0.785) (p < 0.001) for the prediction model of NAFLD remission. CONCLUSIONS: We provide evidence for an association between serum adipsin levels and the remission of NAFLD in a community-based prospective cohort study. Serum adipsin can be a potential biomarker for predicting NAFLD remission.

Topographically Conductive Butterfly Wing Substrates for Directed Spiral Ganglion Neuron Growth.

Spiral ganglion neuron (SGN) degeneration can lead to severe hearing loss, and the directional regeneration of SGNs has shown great potential for improving the efficacy of auditory therapy. Here, a novel 3D conductive microstructure with surface topologies is presented by integrating superaligned carbon-nanotube sheets (SA-CNTs) onto Morpho Menelaus butterfly wings for SGN culture. The parallel groove-like topological structures of M. Menelaus wings induce the cultured cells to grow along the direction of its ridges. The excellent conductivity of SA-CNTs significantly improves the efficiency of cellular information conduction. When integrating the SA-CNTs with M. Menelaus wings, the SA-CNTs are aligned in parallel with the M. Menelaus ridges, which further strengthens the consistency of the surface topography in the composite substrate. The SA-CNTs integrated onto butterfly wings provide powerful physical signals and regulate the behavior of SGNs, including cell survival, adhesion, neurite outgrowth, and synapse formation. These features indicate the possibility of directed regeneration after auditory nerve injury.

Assessment of the effect of deviated nasal septum on the structure of nasal cavity.

The present study was aimed to investigate the effects of DNS on the structure of nasal cavity. The paranasal sinus coronal view CT of 108 patients with DNS and 129 hospitalized patients without DNS was retrospectively analyzed. The transverse diameter of nasal cavity (a), transverse diameter of nasal cavity and paranasal sinus (b), angle between maxillary and palatal bone, interalveolar distance, and maxillary rotation distance were measured. The ratio of a/b in experimental group was 0.367 ± 0.006 which was significantly (P = 0.0023) less than that in control group (0.391 ± 0.005). For the angle between maxillary and palatal bone, there was no significant difference found between DNS and control group for both right and left sides. The interalveolar distance was 40.75 mm in experimental group, and 38.8 mm in control (P = 0.0002). For the maxillary rotation distance, findings were considered as significant (P < 0.0001) in experimental group (11.25 mm) compared with control (10.1 mm). The present study demonstrates that long-term DNS affects the development of nasal cavity and paranasal sinus, as well as increases the interalveolar distance and maxillary rotation distance. These influences may be caused by the alteration of airflow inside the nasal cavities.

Schwannoma of the Sinonasal Tract and the Pterygopalatine Fossa with or without Intracranial Extension.

AIMS: Compared with those in other head and neck regions, schwannomas in the nasal cavity or paranasal sinuses are rare. The aim of this study was to present the experience of the authors in 11 schwannoma cases of the sinonasal tract and pterygopalatine fossa over a decade. METHODS: A retrospective study from 2003 to 2014. RESULTS: Three female and 8 male patients from 22 to 61 years of age (mean age 42 years) were admitted. The most common complaints were unilateral nasal congestion. A total of 10 of the patients received surgery, including 6 functional endoscopic sinus surgeries (FESS). The postoperative course was generally uneventful. Among the patients, 10 remained regionally asymptomatic, and there has been no clinical or radiological evidence of recurrence or residual tumor. CONCLUSION: Surgical treatment is effective for schwannomas of the sinonasal tract and the pterygopalatine fossa with a low recurrence rate. Conducting CT and MRI (particularly fluid-attenuated inversion recovery) before surgery is mandatory. FESS could become the primary treatment of choice.

Emerging biomedical technologies for scarless wound healing.

Complete wound healing without scar formation has attracted increasing attention, prompting the development of various strategies to address this challenge. In clinical settings, there is a growing preference for emerging biomedical technologies that effectively manage fibrosis following skin injury, as they provide high efficacy, cost-effectiveness, and minimal side effects compared to invasive and costly surgical techniques. This review gives an overview of the latest developments in advanced biomedical technologies for scarless wound management. We first introduce the wound healing process and key mechanisms involved in scar formation. Subsequently, we explore common strategies for wound treatment, including their fabrication methods, superior performance and the latest research developments in this field. We then shift our focus to emerging biomedical technologies for scarless wound healing, detailing the mechanism of action, unique properties, and advanced practical applications of various biomedical technology-based therapies, such as cell therapy, drug therapy, biomaterial therapy, and synergistic therapy. Finally, we critically assess the shortcomings and potential applications of these biomedical technologies and therapeutic methods in the realm of scar treatment.

A human-specific cytotoxic neopeptide generated by the deafness gene Cingulin.

Accumulation of mutant proteins in cells can induce proteinopathies and cause functional damage to organs. Recently, the Cingulin (CGN) protein has been shown to maintain the morphology of cuticular plates of inner ear hair cells and a frameshift mutation in CGN causes autosomal dominant non-syndromic hearing loss. Here, we find that the mutant CGN proteins form insoluble aggregates which accumulate intracellularly and lead to cell death. Expression of the mutant CGN in the inner ear results in severe hair cell death and hearing loss in mice, resembling the auditory phenotype in human patients. Interestingly, a human-specific residue (V1112) in the neopeptide generated by the frameshift mutation is critical for the aggregation and cytotoxicity of the mutant human CGN. Moreover, the expression of heat shock factor 1 (HSF1) decreases the accumulation of insoluble mutant CGN aggregates and rescues cell death. In summary, these findings identify mutant-specific toxic polypeptides as a disease-causing mechanism of the deafness mutation in CGN, which can be targeted by the expression of the cell chaperone response regulator HSF1.

Deletion of Luzp2 Does Not Cause Hearing Loss in Mice.

Deafness is the prevailing sensory impairment among humans, impacting every aspect of one's existence. Half of congenital deafness cases are attributed to genetic factors. Studies have shown that Luzp2 is expressed in hair cells (HCs) and supporting cells of the inner ear, but its specific role in hearing remains unclear. To determine the importance of Luzp2 in auditory function, we generated mice deficient in Luzp2. Our results revealed that Luzp2 has predominant expression within the HCs and pillar cells. However, the loss of Luzp2 did not result in any changes in auditory threshold. HCs or synapse number and HC stereocilia morphology in Luzp2 knockout mice did not show any notable distinctions. This was the first study of the role of Luzp2 in hearing in mice, and our results provide important guidance for the screening of deafness genes.

AAV-mediated Gpm6b expression supports hair cell reprogramming.

Irreversible damage to hair cells (HCs) in the cochlea leads to hearing loss. Cochlear supporting cells (SCs) in the murine cochlea have the potential to differentiate into HCs. Neuron membrane glycoprotein M6B (Gpm6b) as a four-transmembrane protein is a potential regulator of HC regeneration according to our previous research. In this study, we found that AAV-ie-mediated Gpm6b overexpression promoted SC-derived organoid expansion. Enhanced Gpm6b prevented the normal decrease in SC plasticity as the cochlea develops by supporting cells re-entry cell cycle and facilitating the SC-to-HC transformation. Also, overexpression of Gpm6b in the organ of Corti through the round window membrane injection facilitated the trans-differentiation of Lgr5+ SCs into HCs. In conclusion, our results suggest that Gpm6b overexpression promotes HC regeneration and highlights a promising target for hearing repair using the inner ear stem cells combined with AAV.

Pcolce2 overexpression promotes supporting cell reprogramming in the neonatal mouse cochlea.

Hair cell (HC) damage is a leading cause of sensorineural hearing loss, and in mammals supporting cells (SCs) are unable to divide and regenerate HCs after birth spontaneously. Procollagen C-endopeptidase enhancer 2 (Pcolce2), which encodes a glycoprotein that acts as a functional procollagen C protease enhancer, was screened as a candidate regulator of SC plasticity in our previous study. In the current study, we used adeno-associated virus (AAV)-ie (a newly developed adeno-associated virus that targets SCs) to overexpress Pcolce2 in SCs. AAV-Pcolce2 facilitated SC re-entry into the cell cycle both in cultured cochlear organoids and in the postnatal cochlea. In the neomycin-damaged model, regenerated HCs were detected after overexpression of Pcolce2, and these were derived from SCs that had re-entered the cell cycle. These findings reveal that Pcolce2 may serve as a therapeutic target for the regeneration of HCs to treat hearing loss.

AAV-Mediated Gene Therapy Restores Hearing in Patients with DFNB9 Deafness.

Mutations in OTOFERLIN (OTOF) lead to the autosomal recessive deafness 9 (DFNB9). The efficacy of adeno-associated virus (AAV)-mediated OTOF gene replacement therapy is extensively validated in Otof-deficient mice. However, the clinical safety and efficacy of AAV-OTOF is not reported. Here, AAV-OTOF is generated using good manufacturing practice and validated its efficacy and safety in mouse and non-human primates in order to determine the optimal injection dose, volume, and administration route for clinical trials. Subsequently, AAV-OTOF is delivered into one cochlea of a 5-year-old deaf patient and into the bilateral cochleae of an 8-year-old deaf patient with OTOF mutations. Obvious hearing improvement is detected by the auditory brainstem response (ABR) and the pure-tone audiometry (PTA) in these two patients. Hearing in the injected ear of the 5-year-old patient can be restored to the normal range at 1 month after AAV-OTOF injection, while the 8-year-old patient can hear the conversational sounds. Most importantly, the 5-year-old patient can hear and recognize speech only through the AAV-OTOF-injected ear. This study is the first to demonstrate the safety and efficacy of AAV-OTOF in patients, expands and optimizes current OTOF-related gene therapy and provides valuable information for further application of gene therapies for deafness.

Identification of two heterozygous deafness mutations in SLC26A4 (PDS) in a Chinese family with two siblings.

OBJECTIVE: To detect genetic cause of two Chinese siblings (patient 1 and 2) with Pendred syndrome. DESIGN: Patients and their parents underwent clinical and genetic evaluations. To identify genetic mutations, sequencing of SLC26A4 was carried out. STUDY SAMPLE: Two siblings and their parents. RESULTS: Clinical evaluations showed that patient 1 suffered from bilateral postlingual progressive sensorineural hearing loss with enlarged vestibular aqueduct and slight diffuse multinodular goiter with euthyroid, and patient 2 suffered from bilateral prelingual progressive sensorineural hearing loss with enlarged vestibular aqueduct and no goiter with euthyroid. Furthermore, the sequence analysis of SLC26A4 indicated that either of the two siblings presented a compound heterozygote for the c.919A>G mutation in the splice site of intron 7 and for the c.1548insC mutation in exon 14. Their mother was a heterozygous carrier of the splice site mutation in intron 7, and their father was a heterozygous carrier of the insertion mutation in exon 14. CONCLUSIONS: Mutation analysis identified a compound heterozygous mutation (c.919A>G/c.1548insC) in SLC26A4 in two Chinese siblings with Pendred syndrome. Also, c.1548insC was first reported in the Chinese population. Although the two siblings from the same family carried the same genotype, they presented different phenotypes.

Primary Surgical Management of Laryngeal Neuroendocrine Neoplasms: A Single Institution Case Series.

Objective: Laryngeal neuroendocrine neoplasms (NENs) are rare diseases. A single institution retrospective study was done of the outcome of patients with laryngeal NENs who undergo primary surgery as the first treatment modality. Methods: Retrospective analysis of medical records of patients with laryngeal NENs between 2009 and 2018. Cases were classified by applying the 2022 World Health organization Classification of Head and Neck Tumors (5th edition). Results: Six patients were eligible at our tertiary center: 1 large cell neuroendocrine carcinoma (NEC), 3 small cell NEC, 1 neuroendocrine tumor grade 1, and 1 neuroendocrine tumor grade 2. All admitted patients received upfront surgeries, including 3 transoral CO2 laser surgeries and 3 total laryngectomies with or without elective neck dissection. Four patients underwent subsequent chemoradiotherapy. Although 3 patients had recurrent disease and distal metastasis, the overall survival was generally improved. Conclusion: According to our institutional experience, upfront surgery in the first-line setting of a multi-modality approach with adjuvant chemoradiotherapy plays a very important role in managing laryngeal NECs, and may confer additional survival benefit in some patients of the large cell carcinoma subgroup.

[Diagnosis and treatment of congenital temporal bone cholesteatoma].

Congenital temporal bone cholesteatoma is a rare lesion in otolaryngology.The disease is locally invasive and may lead to significant complications,including hearing loss（conductive or sensorineural）, temporal bone destruction and intracranial invasion. This article reviews the characteristic symptoms of congenital temporal bone cholesteatoma, testing and imaging of the disease, stage and the current treatment options in order to promote awareness to this rare disease entity and perform early surgical treatment, effectively avoid the destruction of the temporal bone and its surrounding structures, thereby reducing the occurrence of complications. By improving the understanding of the disease and performing early surgical treatment, the destruction of the temporal bone and its surrounding structures can be effectively avoided, thereby reducing the occurrence of complications.

Characteristics of large vestibular aqueduct syndrome in wideband acoustic immittance.

Objective: To describe the characteristics of large vestibular aqueduct syndrome (LVAS) in wideband acoustic immittance (WAI) and to explore whether inner ear deformity has an impact on WAI results. Methods: Subjects with typical LVAS (LVAS group) and control subjects with a normal anatomical structure of the inner ear (control group) were screened from pediatric patients with cochlear implants using thin-slice computed tomography (CT) images of the temporal bone. With inflammation of the auditory canal and middle ear excluded by routine ear examination and 226 Hz acoustic immittance, WAI data were acquired. Then, the maximum absorbance as the major observation indicator on the mean tympanogram was compared between the LVAS group and control group, and a descriptive comparison of the mean tympanogram and frequency-absorbance curve at peak pressure was performed between the two groups. Results: The LVAS group included 21 cases (38 ears), and the control group included 27 cases (45 ears). All LVAS subjects met the Valvassori criteria, and the VA at the horizontal semicircular canal displayed flared expansion. On the mean tympanogram, the maximum absorbance in the LVAS group (0.542 ± 0.087) was significantly higher than that in the control group (0.455 ± 0.087) (p < 0.001). The tympanogram in the LVAS group showed an overall elevation, and the absorbance at all pressure sampling points was significantly higher than that in the control group (p < 0.001). The frequency-absorbance curve at peak pressure first increased and then decreased in both groups, and the LVAS group showed higher absorbance than the control group in the frequency range below 2,828 Hz. The absorbance at 343-1,124 Hz was significantly different between the two groups (p < 0.001), and 343-1,124 Hz was the major frequency range at which the maximum absorbance on the mean tympanogram increased in the LVAS group. Conclusion: Large vestibular aqueduct syndrome (LVAS) shows increased absorbance in low and medium frequency ranges in WAI. The maximum absorbance on the mean tympanogram can serve as a reliable evaluation indicator. Inner ear factors must be considered when middle ear lesions are analyzed by WAI.