Most patients with skull base tumors require radiation therapy as part of their overall treatment, preferably with protons. However, vital and healthy organs, such as the cochlea, are often located in the immediate anatomical vicinity of the tumor. Despite the high precision of the proton beam, irradiating the cochlea is often unavoidable, resulting in an increased risk of hearing loss. To assess the frequency and severity of changes in hearing after proton therapy, we performed a retrospective study in a cohort of 51 patients undergoing proton therapy for skull base tumors. We observed that a hearing threshold shift correlates to the applied radiation dose intensity to the cochlea. In addition, advancing age, hearing sensitivity before proton therapy, and the time elapsed after the end of proton therapy are independently associated with the deterioration of the hearing threshold after proton therapy. These results are essential to adequately inform patients about the treatment’s impact and side effects.p ≤ 0.001), patient’s age , follow-up time , and mean cochlear dose in Gy (RBE)  were all significantly associated with an increase in PTA at follow-up. The applied cochlear dose and baseline PTA, age, and time after treatment were significantly associated with hearing loss after proton therapy.To assess the incidence and severity of changes in hearing threshold in patients undergoing high-dose pencil-beam-scanning proton therapy (PBS-PT). This retrospective cohort study included fifty-one patients ) treated with PBS-PT for skull base tumors. No chemotherapy was delivered. Pure tone averages (PTAs)were determined before (baseline) and after PBS-PT as the average hearing thresholds at frequencies of 0.5, 1, 2, and 4 kHz. Hearing changes were calculated as PTA differences between pre-and post-PBS-PT. A linear mixed-effects model was used to assess the relationship between the PTA at the follow-up and the baseline, the cochlea radiation dose intensity, the increased age, and the years after PBS-PT. Included patients were treated for chordoma (n = 24), chondrosarcoma (n = 9), head and neck tumors (n = 9), or meningioma (n = 3), with a mean tumor dose of 71.1 Gy (RBE) , and a mean dose of 37 Gy (RBE)  was delivered to the cochleas. The median time to the first follow-up was 11 months . The PTA increased from a median of 15 dB (IQR 10.0–25) at the baseline to 23.8 (IQR 11.3–46.3) at the first follow-up. In the linear mixed-effect model, the baseline PTA (estimate 0.80, 95%CI 0.64 to 0.96,  With radiation therapy, skull base tumors, head and neck cancers, and brain neoplasms are challenging to manage, as many critical organs at risk (OARs) are directly near the target volume . For theRadiation-induced hearing loss (RIHL) is a severe adverse effect that significantly decreases the affected patient’s quality of life ,7. The eMost reports on RIHL came from patients treated with photons and indicated an increase, with the total dose >45 Gy for the fractionated photon radiation therapy ,11,12. HAlthough proton therapy is increasingly used worldwide to treat skull base tumors, there is little data on its impact on hearing. This retrospective cohort study aims to assess the frequency and severity of changes in hearing sensitivity after proton therapy of the skull base and to assess potential risk factors for RIHL.This is a retrospective cohort study on patients treated with high-dose pencil-beam-scanning proton therapy (PBS-PT) for skull base tumors at the Paul Scherrer Institute, Switzerland (PSI). The Cantonal Ethics Review board approved the study . From January 2003 to December 2017, 460 patients with a minimum age of 13 years were treated with PBS-PT for skull base tumors. Of this cohort, 51 patients had at least one pre- and one post-treatment audiometry test and were included in the present analysis . Technical aspects of PBS-PT planning have been previously described ,15,16. AThe dose prescription was performed according to the type of tumor, ranging from 52.2 to 77.8 Gy (RBE) (mean 71.1) in 1.8–2.0 Gy (RBE) daily fractions. A generic RBE factor of 1.1 relative to Co-60 was applied, and the dose was expressed in terms of Gy (RBE). None of the patients in this cohort received concomitant chemotherapy.PBS-PT was performed using the pencil beam scanning technique at two scanning gantries at PSI. Single-field uniform dose (SFUD) plans and intensity-modulated proton therapy (IMPT) plans were optimized to achieve the prescribed dose in the tumor while covering at least 95% of the PTV and respecting the dose constraints to the organs at risk (OARs). The dose limit at the cochlea was prescribed with Dmean < 45 Gy, and it was spared as much as possible without reducing target coverage.However, no sparing could be performed in cases where the cochlea was within the CTV or the GTV. In this case, the maximum effort was made to keep the dose to the opposite ear as low as possible. Patients were followed with an MRI and a CT at 3- to 6-month intervals in the first 2–3 years after PBS-PT and annually after that. An annual audiometric test was recommended following PBS-PT and was organized at the discretion of the referring centers. The audiogram results were reviewed and evaluated with two audiologists for all patients included in this study. The evaluated hearing tests before and after proton therapy are the basis for the present study.All patients had a bilateral pure-tone audiometry before starting PBS-PT and at least one audiometry after PBS-PT. A median of 2  follow-up audiometries were performed. One patient was already deaf in one ear during the baseline audiometry; therefore, this ear was excluded from the analysis.Pure tone average (PTA) was calculated for each ear as the average over frequencies of 0.5, 1, 2, and 4 kHz. Higher values of PTA indicate inferior hearing, and an increase in PTA over time suggests a worsening of the hearing threshold. The baseline PTA was classified according to the Global Burden of Disease Expert Group’s (GBDEG) recommendation on hearing loss, in which hearing loss is reported in seven mutually exclusive severity categories . The hearing toxicity of proton therapy was classified using the National Cancer Institute Common Terminology Criteria for Adverse Events (CTC-AE) v. 4.03 grading system. In this grading system, hearing loss is reported as any changes at the frequencies of 1, 2, 3, 4, 6, and 8 kHz during follow-up. Characteristics of patients and their tumors at the baseline, as well as characteristics of the ears affected by tumor at the baseline, and radiation dose to the cochlea, were summarized as median, interquartile range (IQR), and range for most continuous variables or mean; standard deviation (and range for mean radiation dose to the cochlea); and as frequency and percentage for categorical variables. Characteristics at the first follow-up after treatment, available for all patients, were summarized similarly. The distribution of the frequencies of tumor location  concerning PTA was analyzed with the Kruskal–Wallis test, the distribution of tumor localization with cochlear dose with the one-way ANOVA, and the relation of tumor localization to hearing impairment and dose group with a chi-square test.To assess whether the change in PTA between the baseline and the first follow-up (mean 11 months) after treatment was significant, we performed a nonparametric Wilcoxon signed-rank test between the two paired samples.p-values (testing the null hypothesis of each coefficient being zero). Statistical analyses were performed in the R system for statistical computing, version 4.0.4 (R Core Team 2021) [To assess the association of the radiation dose to the cochlea on the PTA during follow-up, we used the full, longitudinal data of all PTAs on both ears for each patient, including all post-treatment audiometries  per patient, which were performed at irregular time intervals . To account for the hierarchical nature of these data, we used a linear mixed-effects model with a random intercept per ear nested within a patient. PTA at the baseline, patient age in years (at the respective follow-up), years since treatment, and mean dose to the cochlea were used as explanatory variables. To account for the serial autocorrelation between repeated measurements, a continuous, autoregressive process, i.e., an AR (1) process for the continuous-time covariate years since treatment, was used. Age at the follow-up was used instead of age at the baseline to model a separate slope for increasing age and increasing time since treatment, assessing whether the PTA changed more rapidly after treatment than due to increasing age. Coefficient estimates from this model are reported together with 95% confidence intervals (CI) and am 2021) .Between the baseline audiometric test and the start of proton therapy, there was a median of 17 days (IQR 8.5–35), and between treatment start to the first follow-up audiometric test, there was a median of 11 months (IQR 5.5–33.7), starting from the beginning of PBS-PT. Further analyses were performed on data from all ears of the 51 patients. The one deaf ear at the baseline was excluded, and a total of 101 ears (cochleas) were therefore analyzed.The median baseline PTA for all ears was 15 dB (IQR 10–25), and grading according to the GBD classification revealed that 17/101 ears (16.9%) already had moderate to profoundly poor hearing (35–95 dB) .p < 0.001). Additionally, the ipsilateral cochlear dose of lateralized tumors was higher than in both cochleas in midline tumors (59.4 Gy vs. 37.1. Gy (RBE))  (SD 22.5). Patients with unilaterally localized tumors had a significantly higher mean dose on the ipsilateral cochlea (59.4 Gy (RBE), SD 16.4) than on the contralateral side (13.4 Gy (RBE); SD 12.29; y (RBE)) .p < 0.001). This impairment was more pronounced in the ipsilateral ears of patients with lateralized tumors (32.5 dB HL) than in patients with midline tumors (28.9 dB HL) (The median PTA increased significantly by 8.7 dB from 15 dB HL at the baseline to 23.7 dB HL (IQR 11.3–46.3) at the first follow-up, indicating an impairment of hearing sensitivity (9 dB HL) .p = 0.047) (In 82/101 (81.2%) ears, the baseline hearing test also included information on bone conduction, which determined sensorineural (24.5%) and mixed (6.9%) hearing disorders as the most common types. At the first follow-up, the percentage of sensorineural disorders was similar (26.7%) to the baseline, but there were significantly more mixed hearing disorders (15.8%) (= 0.047) .According to the CTC classification, 16 patients (31%) had an unchanged hearing sensitivity at the first follow-up visit, 11 (22%) patients presented with mild hearing loss of 15–25 dB (CTCAE Grade 1), and 24 patients (47%) presented with moderate to severe hearing loss of >25 dB (CTCAE Grade ≥ 2), respectively . Most heThe linear mixed-effects model  estimateOur data show that patients requiring PBS-PT for skull base tumors are at significant risk for hearing loss. In addition to the applied radiation dose to the cochlea, we identified baseline PTA, age at follow-up, and time after the end of PBS-PT as independent risk factors for hearing loss after proton therapy. The clinical effects of ionizing radiation on hearing have been studied mainly with photons. To the best of our knowledge, this is the first investigation to address the impact of PBS-PT on the hearing function in patients with skull base tumors. In our study, a gradual relationship was observed between the applied cochlear dose and the deterioration of hearing sensitivity, measured as PTA: each additional Gy to the cochlea resulted in a 0.34 dB increase in hearing loss. Interestingly, the changes in hearing were also seen at doses below 32 Gy (RBE), which is significantly lower than the cochlea dose constraints mentioned in the literature ,21,22,23Previous studies recommended limiting the cochlea’s mean dose to ≤45 Gy, with conventional fractionation ,20,23. IFollowing these common recommendations, the treatment policy at PSI is always to strive for the dose to the cochlea to be below 45 Gy (RBE). However, if the macroscopic tumor is next to or even abutting the cochlea, it is impossible to maintain this dose constraint, even with advanced PBS-PT techniques. In the present study, this was the case in 20 skull base tumors lateralized to one side, where a median dose to the cochlea of 58.8 Gy (RBE) was applied. However, in these situations, special care was taken to keep the dose to the opposite cochlea as low as possible (mean 13.4 Gy) to preserve hearing function in at least one ear see . In patiWe also observed that advancing age leads to a hearing loss of 0.30 dB/year, independent of the radiation dose to the cochlea. This result is in line with a recent study on healthy subjects that indicated a progression of hearing loss of 0.29 and 1.35 dB/year (low and high frequencies) in older adults independent of clinical and socioeconomic factors . In contCategorizing adverse events after radiotherapy according to the CTC criteria has become widely established as a measurement method for recording. In the present study, 26% of patients had CTC hearing toxicity grades 1 and 2, and 43% had ≥Grade 3 toxicity. However, CTC classification includes hearing changes across all frequencies, even high frequencies essential for daily speech use in noisy surroundings. Thus, CTC classification reflects the true clinical picture from the patients’ perspective for daily life communication. On the contrary, the PTA evaluation uses only those frequencies , which are significant for speech understanding when quiet . In the An exciting aspect of our study is that none of the included patients received chemotherapy. This is where our study differs from others, in which primarily concomitant chemotherapy was given, which is a contributing factor for ototoxicity ,20.As with all retrospective analyses, this study has potential limitations, including but not limited to uncontrolled patient selection. We performed multivariate analyses to eliminate potential confounders when estimating the association of the mean dose to the cochlea with hearing sensitivity during follow-up, but some residual confounding likely remains. Additionally, the number of cases is small and might lead to non-representative results for a larger patient cohort.In summary, we have shown that the baseline PTA, cochlear dose, years after proton therapy, and age at follow-up have independent effects on hearing loss after PBS-PT. Therefore, we believe it is impossible to define a safe dose to the cochlea that will reliably prevent hearing loss after PBS-PT. This fact should be understandably explained to patients so they are sufficiently informed to give informed consent for radiation.


Hearing is one of the main ways with which one person can contact the external world; it plays a key role in their integration with society.The objective of this study was to analyze the results of the hearing, medical and genetic evaluation of high-risk infants who failed the newborn hearing screening.Clinical and experimental study. We assessed thirty-eight neonates, with ages between one and six months. The infants underwent the following procedures: medical interview; immittance testing; Brainstem Auditory Evoked Potential; Transient Evoked Otoacoustic Emission and otorhinolaryngological evaluation. DNA extraction from the oral mucosa was performed for genetic studies using the protocol method adapted from the Human Genetics Lab of the CBMEG/UNICAMP.Regarding gender and presence of risk factors, significant statistically differences were not found in normal hearing infants and in those with hearing loss. Concerning gestational age, term infants were more affected by hearing loss. Hearing loss was identified in 58% of the sample, conduction hearing loss represented 31.5% (12/38) and neurossensory 28.9% of cases. There were none of the genetic mutations most commonly seen in cases with a genetic etiology.Hearing loss was identified in the majority of High-risk infants. Hearing is one of the main forms by which individuals establish contact with the external world, and is thus of utmost importance for integration within society.The estimated incidence of significant and bilateral hearing loss is one to three for each thousand newborns at low risk for hearing lossUniversal neonatal hearing screening has been recommended as the main strategy to reduce the age at which a diagnosis of hearing loss may be madeA test battery with behavioral and electrophysiologic tests is recommended for the diagnosis of hearing loss, such as: observing auditory behavior relative to calibrated and non calibrated sounds, acoustic immittance testing, otoacoustic emissions, and brainstem auditory evoked potential (BAEP). These suggested tests help define the type, grade, and configuration of hearing loss, which result in a differential diagnosis, interventions, and the indication of hearing aids for sensorineural, mixed, or conductive hearing lossThere are several causes of congenital hearing loss – those acquired during the prenatal period or within the first days after delivery. The causes of hearing loss may be genetic or environmental. The etiology of genetic origin hearing loss may be syndromic, those associated with craniofacial or neck malformations, skeletal dysplasia, skin or ocular abnormalities, neurologic diseases, renal or metabolic dysfunction, and others. It has been estimated that 30% of cases of prelingual deafness are syndromic and 70% are non-syndromicEnvironmental factors include congenital infections, perinatal, and postnatal factors.Thus, the purpose of this study was to investigate the results of an audiologic, otorhinolaryngologic, and genetic evaluation of high-risk infants that failed in neonatal hearing screening, taking into account the variables male/female sex, number of risk indicators, and gestational age. Hearing loss was also analyzed according to type, grade, affected side, and possible etiology.A cross-sectional contemporary cohort study was carried out. It was approved by the institutional review board of the Research Ethics Committee of the School of Medical Sciences of the University of Campinas (FCM/ UNICAMP). The reference number was 028/2008.All infants born at the Women's Hospital - Prof. Dr. José Aristodemo Pinotti – CAISM/UNICAMP that remained in the neonatal ICU and that failed hearing screening, from February 2009 to March 2010 were enrolled. Infants born in other hospitals or that did not undertake all the evaluations within the study period were excluded. There were 52 infants sent for a diagnostic assessment; this number is close to the sample size based on the mean monthly delivery number at the hospital (250 deliveries) and the number of newborns that are admitted into the ICU (40 per month). Thus, there were 500 newborns in 12 months, of which on average 10% (50 infants) failed in hearing screeningA staff speech therapist of the hospital Audiology Diagnostic Laboratory carried out the audiological evaluation. The infants were aged 1 to 6 months when tested; the procedures were as follows: a clinical history, evaluation of the middle ear status, BAEP testing (electrophysiologic threshold and auditory pathway integrity), and transient otoacoustic emissions (TOAE). Infants slept naturally while the procedures were applied.The clinical history was taken from family members to gather the following data: identification, the hospital discharge report, and information about motor, auditory and language development. The JCIHThe electrophysiologic threshold and auditory pathway status were assessed by BAEP using an Eclipse EP 25 Interacoustics device with in-ear phones. Auditory pathway status was investigated with non-variable 80 dB clicks presented 19 times per second, which make it possible to evaluate auditory pathways and identify issues up to the brainstem. The electrophysiologic threshold was gathered by sloping stimuli until reaching the lowest stimulus intensity that caused the V wave to appear. The stimulus was applied twice to obtain a reproducible tracing and make sure the response was present. Surface electrodes were attached over the right and left mastoid and the frontoparietal regions of infants after cleaning the skin with abrasive paste and applying electrolytic paste. The following parameters were assessed: presence of waves I, III, and V; absolute wave I, III, and V latencies; I-V, I-III, and III-V interpeak latencies; wave V amplitude relative to wave I amplitude; and I-V interpeak or wave V latency interaural difference. TOAE were measured by an ILO 292 USBII device.The tympanometric curve was used to evaluate the middle ear; the probe tone was set at 1,000 Hz and the ipsilateral acoustic reflex was measured from 500 to 4,000 Hz. A 235 H, Interacoustics device was used.Responses in each test were recorded on answer sheets. Normal hearing was considered as a click electrophysiologic threshold under 30 dB, absolute and interpeak latencies within expected values for the gestational ageInfants with abnormal results in at least one auditory test were sent to an otorhinolaryngologist to be evaluated. One of the researchers supervised this assessment, which consisted of otoscopy to examine the outer ear canal and the tympanic membrane, and imaging if needed.GJB6-D13S1830) and D(GJB6-D13S1854) deletions with previously reported primersMTRNR1 gene to detect the A1555G mutation; the abovementioned primer pairs were used. Restriction analysis to detect mutations was applied to amplification products.Hearing was classified as normal or impaired depending on the analysis of the audiologic and otorhinolaryngologic assessments. Hearing loss was classified according to the typeThe statistical analysis was made with the SAS software version 9.1.3. The significance level was 5%, and was indicated by an asterisk (*).There were 52 infants that failed hearing screening and that were referred for diagnosis. Of these, 38 infants (73%) participated in the study and underwent the full diagnostic process. The remaining infants either did not come to scheduled visits or failed to sleep after BAEP testing, among other factors, thereby not undergoing the full diagnostic procedure within the study period.GJB2) gene was not encountered, neither were the D(GJB6-D13S1830) and Δ(GJB6-D13S1854) deletions on the GJB6 gene and the A1555G mutation on the MTRNR mitochondrial gene.The 35delG mutation on the conexin 26  were male and 50% were premature. Most of the children were male, which reflects the demography of most neonatal ICUsAll infants had at least one risk indicator in the clinical history .There was no statistically significant difference in normal and impaired hearing neonates in relation to the variables gender and number of risk indicators. On the other hand, term neonates were more affected by hearing loss than premature infants, and this finding was statistically significant . Term neAnalysis of the tests showed that 42% (16/38) of children had normal results in all tests . Their fMostly bilateral conductive hearing loss was present in 31.5% (12/38) of the sample . Conductth and 12th months, and occur least in the first three months of lifeBone et al.Preventive measures, such as encouraging breast feeding, positioning the infants adequately while nursing, avoiding passive smoking, and other initiatives, may minimize conductive hearing losses. Such losses impair the development of hearing and language by causing sensory deprivation fluctuating hearing that is typical of otitis media.The incidence of hearing loss in Down syndrome infants ranges from 2.6% to 67.5%; it has been attributed mainly to a high rate of secretory otitis mediaThese infants were receiving otorhinolaryngological treatment, periodic audiologic evaluations, and were monitored for language and hearing development. Published reports have shown that some cases of middle ear involvement resolve spontaneously without harm to development, whereas in other cases, the disease becomes chronic. In this case it may compromise language and educational development, and if left untreated, may progress and involve the mastoid cells or even the cranial cavity, which leads to serious complications including inner ear involvement and sensorineural hearing lossWe found sensorineural hearing loss in 28.9% (10/38) of the sample; five cases were moderate to severe, and five cases were profound loss . These iThe results of three infants among the cases of bilateral profound sensorineural hearing loss were compatible with the auditory neuropathy spectrum disorder (ANSD), which consist of cochlear microphonism, absence of waves I, III, and V at 100 dB in BAEP testing, TOAE, a type A tympanometric curve, and absence of acoustic reflexes. An elevated bilirubin level was a risk indicator in the three cases – bilirubin levels were above 28 mg/dL.th cranial nerve and/or brainstem dyssynchrony, disordered inner hair cells, dysfunctional spiral ganglion fibersth cranial nerveFindings in the ANSD include 8(GJB2) mutation. The V27I polymorphism on the GJB2 gene was found in subject 1 in heterozygosis, which is not relevant for hearing loss, as is the case for silent mutations. Although the 5delG mutation was not found in the sample after screening, it is extremely important since it is present in 70% of cases of deafness when the GJB2 gene is involved. The prevalence of carriers of the 35delG mutation in Brazil, as encountered in a survey of 620 neonates in Campinas (Sao Paulo state) is 0.97% – about 1:103 heterozygotesGJB2 gene reduces the empirical risk of a genetic cause of deafness. Besides the GJB2 gene, Δ(GJB6-D13S1830) and Δ(GJB6-D13S1854) deletions on the GJB6 gene were also analyzed. None of our subjects had any of these deletions. We also tested for the A1555G mitochondrial mutation, which is associated with hearing loss and use of aminoglycoside antibiotics, and did not find this abnormality in any of our study subjects.All study subjects were normal for the 35delG conexin gene 26 Declau et al.According to the literature, the cause of hearing loss is genetic in 50% of cases and environmental in another 50% of cases in developed countriesIn our study, environmental factors in the history of infants were the most likely causes, because no genetic mutations commonly implicated in hearing loss were found . IdentifAnalysis of audiologic, otorhinolaryngological, and genetic findings in high risk nursing infants that failed hearing screening showed that the frequency of hearing loss was higher in term neonates compared to premature neonates. Children with predominantly bilateral conductive and sensorineural hearing loss had a similar distribution. The likely causes of sensorineural hearing loss are environmental factors. The diagnosis and etiology of deafness are extremely important for establishing the prognosis and appropriate therapy.


Despite the enormous efforts made to develop other fusion inhibitors for HIV, the enfuvirtide (known as T20) peptide is the only approved HIV-1 inhibitory drug so far. Investigating the role of potential residues of the T20 peptide’s conformational dynamics could help us to understand the role of potential residues of the T20 peptide. We investigated T20 peptide conformation and binding interactions with the HIV-1 receptor  using MD simulations and docking techniques, respectively. Although the mutation of E143 into alanine decreased the flexibility of the E143A mutant, the conformational compactness of the mutant was increased. This suggests a potential role of E143 in the T20 peptide’s conformation. Interestingly, the free energy landscape showed a significant change in the wild-type T20 minimum, as the E143A mutant produced two observed minima. Finally, the docking results of T20 to the gp41 receptor showed a different binding interaction in comparison to the E143A mutant. This suggests that E143 residue can influence the binding interaction with the gp41 receptor. Overall, the E143 residue showed a significant role in conformation and binding to the HIV-1 receptor. These findings can be helpful in optimizing and developing HIV-1 inhibitor peptides. The human immunodeficiency virus (HIV) is a causative agent for acquired immunodeficiency syndrome (AIDS) infection, a chronic and life-threatening disease. HIV utilizes its envelope glycoproteins to initiate the entry process into host cells . SpecifiThe enfuvirtide (T20) peptide was the first member of HIV-1 entry inhibitors granted FDA approval for clinical use to treat HIV infection, which occurred in 2003 ,5. The TDespite the enormous efforts to develop other fusion inhibitors for HIV, the T20 peptide is the only approved HIV-1 inhibitory drug so far. The challenges of developing new HIV inhibitors are attributed to several factors such as the high mutation rate of HIV envelope proteins  and lack of knowledge regarding the exact mechanism of action of the T20 peptide. Moreover, the glycosylation pattern of gp41 envelope proteins act as a “glycan shield” for the conserved  regions [Molecular dynamics (MD) simulations is a powerful tool for studying biomolecular structures as well as identifying conformational-dependent residues in proteins . MoreoveThe C-α root mean square deviation (RMSD) method was used to assess the stability of the MD simulation systems. The WT T20 and E143A mutant systems showed a plateau convergence after 50 ns of the MD simulations’ run A. This iThe radius of gyration (Rg) was used to assess the conformational compactness and the size of the proteins . TherefoPrincipal component analysis (PCA) was used to identify major contributing components to the protein dynamics ,15. It iThe free energy landscape (FEL) of the all-peptide atoms was used to evaluate conformations at minima during the MD simulations . The FEL200ns) and the E143A (E143A200ns) mutant were selected as a representative dynamic structure for each MD simulations system. Afterward, both the T20200ns and E143A200ns structures were docked to the X-ray structure of the gp41 receptor using multiple docking techniques HADDOCK and HawkDock servers. There was agreement in the docking results between both techniques especially in the first ranking models of HawkDock using MM-GBSA scoring  T20 peptide was obtained from the Protein Data Bank (PBD ID 5ZCX) The mutation of the T20 peptide was performed using PyMOL to obtain the E143A mutant [eviously ,12.MD simulations for the WT T20 and E143A systems were performed using the GROMACS 5.1.4 program with the CHARMM27 force field . The X-rThe online HADDOCK2.4 and HawkDock servers were used to dock the WT T20 peptide or the E143A mutant to their gp41 receptor (PDB ID 2OT5) ,22. For The T20 peptide was the first member of the HIV-1 inhibitor class granted FDA approval. Investigating the potential residues in T20 might help us to overcome the current challenges faced in developing new inhibitor drugs for HIV-1. Our MD simulations study showed that the E143 residue of the T20 peptide played a significant role in the conformational structure of the peptide. The E143A mutation not only affected the conformations of the T20 peptide but also the binding interaction with the HIV-1 receptor . Although we conducted 200 ns long MD simulations, a longer simulation time might be more informative regarding peptide dynamics. Moreover, experimental studies might confirm these findings using structural instruments such as nuclear magnetic resonance (NMR) or surface plasmon resonance (SPR) studies.


Additional genetic testing revealed pathogenic copy number variants in the STRC gene in two families. In summary, regular re-evaluation of hearing loss patients with presumably genetic etiology after negative molecular diagnostics is recommended, as we might miss newly discovered deafness genes. The switch from gene panel analysis to whole exome sequencing or whole genome sequencing for the testing of congenital hearing loss seems promising.Congenital hearing loss has an impact on almost every facet of life. In more than 50% of cases, a genetic cause can be identified. Currently, extensive genetic testing is available, although the etiology of some patients with obvious familial hearing loss remains unknown. We selected a cohort of mutation-negative patients to optimize the diagnostic yield for genetic hearing impairment. In this retrospective study, 21 patients (17 families) with negative molecular diagnostics for non-syndromic hearing loss  were included based on a positive family history with a similar type of hearing loss. Additional genetic testing was performed using a whole exome sequencing panel (WESHL panel v2.0) in four families with the strongest likelihood of genetic hearing impairment. In this cohort ( Hearing impairment is one of the most common sensory defects in children . Based oThe etiology of hearing loss is diverse. A genetic cause is presumed or identified in more than 50% of cases. About 25% of cases of congenital hearing loss are acquired, and less than 25% are idiopathic ,7. AcquiThe therapeutic options for hearing loss include conventional hearing aids, cochlear implants, and adapted educational needs. Conventional hearing aids are successfully used in most patients with mild to severe sensorineural hearing loss. However, for patients with severe to profound sensorineural hearing loss, a cochlear implant is usually preferred . FindingGJB2/GJB6 screening. Gene panels are regularly updated based on recent knowledge, and a transition from custom targeted panel testing to exome sequencing with a virtual panel has been introduced recently. Unfortunately, even after a comprehensive etiological work-up, the cause of hearing loss is not discovered in a considerable proportion of patients [Given the prevalence of genetic hearing loss, molecular testing in an early stage is recommended. Technologic innovations in genetic research have expanded our knowledge on genetic hearing loss tremendously during the past decades. Where in early years single genes were tested sequentially, in present times a syndromic and/or non-syndromic test panel is widely implemented, whether or not it is preceded by patients .This article aimed to describe a cohort with negative molecular diagnostics for non-syndromic hearing loss with a strong likelihood of a genetic cause based on an obvious familial history for the same type of hearing loss. Furthermore, for some of those patients, we aimed to explain why no etiological diagnosis was found, and proved our hypotheses by additional genetic analyses. In addition, the management and future possibilities for genetic testing of patients with negative molecular diagnostics for non-syndromic hearing loss will be discussed.A combination of a retrospective study and literature study was performed. Additional testing with an updated gene panel was performed in some patients after approval of the respective families. The study was approved by the Ethical Committee of Ghent University Hospital, Belgium.n = 21) have been selected from the database of the otogenetics consultation of the otorhinolaryngology department of Ghent University Hospital, Belgium. Patients in whom no pathogenic mutation had been identified by a previous molecular analysis  were selected by a group of otorhinolaryngologists and geneticists of Ghent University Hospital based on a very high likelihood of having a genetic cause for their hearing loss. This likelihood was mainly based on an obvious familial history for the same type of hearing loss. Patients with arguments for a non-genetic cause of hearing loss  were excluded.Patients included in this article , of whom seven agreed.GJB2/GJB6 genes using Sanger sequencing by the Center for Medical Genetics Ghent, Belgium. A gene panel analysis was performed by SBS sequencing technology  after Haloplex enrichment of a gene panel of genes causing hearing loss. Different versions of the non-syndromic deafness gene panel  have been used as panel testing for those patients in the past. The retrieved variants were reported based on five classes depending on their likelihood to be pathogenic according to the recommendations of the American College of Medical Genetics and Genomics (ACMG) and the Association for Molecular Pathology (AMP) [All of the 21 included patients underwent genetic testing using the targeted gene panel for non-syndromic hearing loss at the Center for Medical Genetics, Antwerp, Belgium. In earlier years, this test was preceded by the exclusion of mutations in the gy (AMP) . All patSTRC gene were confirmed via STRC-specific long-range PCR followed by a sequence analysis of the relevant STRC coding exons. Analysis for STRC copy number variants was performed using sequencing data and copy number loss was confirmed by multiplex ligation-dependent probe amplification (MLPA) analysis with the P461-A1kit .Of the included patients, seven underwent whole exome sequencing conducted via SBS sequencing technology  after enrichment with the Twist Human Core Exome kit with additional human RefSeq transcripts and the mitochondrial genome . The 146 genes included in the WESHL panel v2.0 were analyzed for variants with JSI SeqPilot software v5.3.3  . In addiSequence data were analyzed with SeqNext analysis software  against the Hg19 exome build reference sequence. For all individual genes a minimal 30× coverage was obtained for more than 95% of the coding sequences, and for the total gene panel a minimal 30× coverage was obtained for more than 98% of the coding sequences. A minimal minor allele frequency threshold of 15% was used for variant detection.After the selection of patients, a database was created in Microsoft Excel . This database included general information about the patients , data on the type and etiology of hearing loss, severity, onset, type, symmetry and audiometric configuration of hearing loss, familial history of hearing loss, cytomegalovirus infection status, and results of molecular testing with the non-syndromic deafness gene panel of the Center for Medical Genetics Antwerp (DOOF_v5_NS—DOOF_V11_NS).These data were obtained from the electronic health record of the patients. Severity of hearing loss was classified into six categories ((slight (16–25 decibel hearing level (dB)), mild (26–40 dB), moderate (41–55 dB), moderately severe (56–70 dB), severe (71–90 dB) or profound (≥90 dB)) . For asyDifferent databases  were used to find relevant publications. The reference list of the most important publications was used to search for essential missing publications. EndNote 20  was used as the citation manager.The selected study population included 21 patients, of whom 16 were male and 5 were female. Their ages at inclusion ranged between 4 and 13 years old, with the majority born between 2015 and 2018 (15 patients). All included patients had bilateral hearing loss. Fifteen of them had symmetrical hearing loss, whereas six had asymmetrical hearing loss. The hearing loss severity of the included patients can be found in The targeted gene panel for non-syndromic hearing loss, performed in all patients, resulted in a total of 65 variants in 39 different genes . Nine ofEight patients out of four families were selected for additional genetic testing based on the strongest familial history for the same type of hearing loss. Seven of them agreed to perform additional testing. The pedigrees of the four families are depicted in In this study, patients with presumable hereditary hearing loss and negative molecular testing in the past have been investigated. We found most patients exhibiting moderate and moderately severe hearing loss (71%). Patients with profound hearing loss seem underrepresented compared with the general distribution of congenital hearing loss severity. In general, in more severe forms of hearing impairment the cause is more frequently found than in milder degrees of hearing loss . This suAsymmetric hearing loss was present in 24% of our cohort. Sloan-Heggen et al.  reportedGenetic variants were found in more than 40 different genes in the patients of the study cohort. To date, more than 120 genes are identified as causing non-syndromic hearing loss . Custom STRC gene has been shown to be the most commonly mutated gene in patients with this type of hearing impairment. STRC causes hearing loss in an autosomal recessive manner [STRC gene sequence data are difficult to interpret due to the existence of an almost identical pseudogene pSTRC [STRC gene was only recently (March 2020) added to the non-syndromic hearing loss panel used in the center for Medical Genetics in Antwerp. Based on a strong family history of hearing loss and the audiograms showing moderate to moderately severe hearing loss, a subset of patients with no molecular diagnosis was recontacted to perform an updated deafness gene panel containing the most recent deafness genes. More specifically, eight patients of four different families were recontacted, of whom seven agreed to participate. The main goal was to identify the molecular causes of hearing loss in additional deafness genes, and in particular in the recently added STRC gene. In half of the families (four out of seven patients), STRC pathogenic variants were found, some in cis with a CATSPER2 deletion. The latter is a gene accounting for sperm motility. Deletions in this gene often go hand in hand with deletions in the STRC gene [Of the included patients, 52% presented with moderate sensorineural hearing loss. The e manner ,17. The ne pSTRC . The STRTRC gene . This geTRC gene ,20.CDH23, MYO15A and PTPRQ genes were mainly detected. Given the existence of digenic inheritance, it does not imply that a heterozygous variant in an autosomal recessive deafness gene is not involved in hearing loss. True digenic inheritance occurs when two non-allelic mutations on two separate genes are necessary and sufficient to cause disease [CDH23 and ATP2B2 genes and of variants in the SLC26A4 gene and FOXI10 or KCNJ10 genes has been suggested [In addition to the detected disease-causing variants, we observed variants of uncertain significance (VUS) in several deafness genes. Variants in the  disease . Digenicuggested ,23,24. Huggested . In geneuggested , and thiWith these results in mind, we see several opportunities to improve the diagnostic yield for genetic hearing impairment. Different genetic testing strategies can be used to detect genetic alterations that can cause hearing loss. Currently, next generation sequencing custom targeted gene panel testing is the gold standard for the genetic analysis of hearing loss in most centers. There are other ways to establish an etiological diagnosis, however. Three commonly used testing strategies  all have their own advantages and disadvantages, which we summarized in n = 21). One of the largest disadvantages of this testing strategy is that only known deafness genes are included and that it is very static, as it is not easy to change these panels. A transition towards exome or even genome sequencing is becoming the gold standard. Exome sequencing with the use of virtual panels to restrict the analysis to specific genes related to a specific disorder using bioinformatic filtering is an increasingly favored approach for genetic testing. This technique has several advantages compared to the targeted approach. First, there is less chance of secondary findings compared to exome or genome sequencing without a virtual panel, thanks to the fact that only a panel of genes associated with hearing loss is analyzed. It also leads to less detection of variants with uncertain significance, which are often difficult to interpret and can cause uncertainty for both patients and clinicians. The technique is very flexible because genes can easily be added to and removed from the panel when new genetic knowledge becomes available. Even a retrospective analysis of novel deafness genes is possible without new blood sampling, again stressing the importance of the regular re-evaluation of patients [Initially, custom targeted gene panel testing was performed in this study population (patients ,28,29,32Genetic variants are mostly classified into five classes based on the criteria of the ACMG-AMP . In receThis study has a few limitations. First, the study population only included 21 patients. In addition, the study is retrospective in design. There is also a selection bias because patients were not randomly selected, but were selected by an expert committee to be the most likely having genetic hearing loss. Minor information bias is also possible as the database is based on the patients’ electronic health records and different caregivers sometimes have a different way of interpreting clinical information.STRC seems to be a prevalent cause of hearing loss. In patients with previous negative molecular diagnostics for non-syndromic, mild to moderately severe hearing loss, the STRC gene should be analysed in case it was not performed in the past.In summary, clinical and audiometric re-evaluation combined with updated genetic testing can be successful in establishing an etiological diagnosis in some cases without a molecular diagnosis at first. The implementation of whole exome or whole genome sequencing with a virtual panel as the gold standard for genetic testing in hearing loss should be considered, instead of custom targeted gene panel testing.


Gliricidia sepium (Jacq.) Walp. adapts to a very high level of salt stress (≥20 dS m−1) and resumes the production of new leaves around 2 weeks after losing all leaves due to abrupt salinity stress. The integration of metabolome and transcriptome profiles from gliricidia leaves points to a central role of the phenylpropanoid biosynthesis pathway in the short-term response to salinity stress. In this study, a deeper untargeted metabolomics analysis of the leaves and roots of young gliricidia plants was conducted to characterize the mechanism(s) behind this adaptation response. The polar and lipidic fractions from leaf and root samples were extracted and analyzed on a UHPLC.ESI.Q-TOF.HRMS system. Acquired data were analyzed using the XCMS Online, and MetaboAnalyst platforms, via three distinct and complementary strategies. Together, the results obtained first led us to postulate that these plants are salt-excluding plants, which adapted to high salinity stress via two salt-excluding mechanisms, starting in the canopy—severe defoliation—and concluding in the roots—limited entry of Na. Besides that, it was possible to show that the phenylpropanoid biosynthesis pathway plays a role throughout the entire adaptation response, starting in the short term and continuing in the long one. The roots metabolome analysis revealed 11 distinct metabolic pathways affected by salt stress, and the initial analysis of the two most affected ones—steroid biosynthesis and lysine biosynthesis—led us also to postulate that the accumulation of lignin and some phytosterols, as well as lysine biosynthesis—but not degradation, play a role in promoting the adaptation response. However, additional studies are necessary to investigate these hypotheses.The multipurpose tree  It is one of the most well-known multipurpose trees, known for its ability to adapt very well to a wide range of soils, from eroded acidic soils, sandy soils, heavy clay, limestone, and alkaline soils  analysis.The current study is a follow-up to our previous study  Steud.] used in this study belongs to the Gliricidia Collection at Embrapa Tabuleiros Costeiros (www.embrapa.br/en/tabuleiros-costeiros). After soaking the seeds in 2% sodium hypochlorite and Tween® 20 for 5 min under slow agitation, we washed them with sterile water and dried them on sterilized filter paper. Then they were placed in a Petri dish with filter paper moistened with sterilized water until the radicle emission. Subsequently, each germinated seed was transferred individually to a 5 L plastic pot  containing 4 kg of substrate previously prepared by mixing sterile soil, vermiculite, and a commercial substrate (Bioplant®), in the ratio 2/1/1 (v/v/v); and kept in a greenhouse for 3 months.The accession of gliricidia  + m/z 350.11867 and [M-H]—m/z 348.10288) was added to each sample as an internal standard for peak normalization.A high-resolution mass spectrometry equipment (HRMS) , coupled to the UHPLC system, performed the chemical mass to charge ratio (m/z) detection using an electrospray source in positive [ESI (+)—MS] and negative [ESI (–)—MS] ionization modes. Final plate offset, 500 V; capillary voltage, 3,800 V; nebulizer pressure, 4 bar; dry gas flow, 9 L minm/z 70–1,000 (polar fraction) and m/z 300–1,600 (lipidic fraction); spectrum rate, 3 Hz; ionic, positive polarity; pre-pulse storage, 8 μs; funnel 1 RF, 250.0 Vpp. The UHPLC-MS and UHPLC-MS/MS data were acquired by HyStar Application version 3.2 .Tandem mass spectrometry (MS/MS) parameters have been adjusted to improve mass fragmentation, with collision energy ranging from 20 to 50 eV, using a step method. Precursor ions were acquired using the 3.0 s cycle time. The general AutoMS settings were: mass range, t-test (Welch t-test) was used for statistical analysis.The raw data from UHPLC-MS were exported as mzMXL files, using DataAnalysis 4.2 software . During data pre-processing, peak detection, retention time correction, and metabolites alignment were performed using XCMS Online , and all data variables from the lipidic fraction were also normalized by internal standard . Prior to the multivariate analysis, no data transformation was applied to the data matrices containing m/z and intensities values. All three sets of data were scaled using the Pareto scaling, which reduced the relative importance of large values, keeping data structure partially intact, and although sensitive to large fold changes, it stays closer to the original measurement than Autoscaling.The processed data (csv file) were exported to MetaboAnalyst 5.0, and submitted to analysis in the Statistical Analysis module  ≤ 0.050, of the Welch t-test. The selected DEPs were then submitted to analysis in the MS Peaks to Pathway module  were selected according to the following criteria: adjusted In the case of a DEP with two or more matched forms (isotopes) and later a matched compound with two or more DEPs, the criterion of metabolite selection applied was the mass difference comparing to the metabolite database—choosing the smallest one. Then, we used the formula and exact mass data from KEGG to perform the putative annotation of the metabolites of interest, with just one candidate on each detected ion.A. thaliana pathway library as reference metabolome (from October 2019).The KEGG IDs of the matched compounds were then submitted to pathway analysis  and visualization in the Pathway Analysis module .Initially, correlation analyses were done between pairs of differentially expressed metabolites—a pairwise combination of the different scenarios evaluated—using homemade python scripts. The input data for the correlation analysis was the Log2(FC) of the metabolites and an enrichment analysis was initially carried out to discover the metabolic pathways to which they belong. The “KEGG feature distribution” module was used, which showed the metabolic pathways and the amount of metabolites differentially expressed in each pathway. To verify which were these metabolites, for each pathway, the module “Map data on the KEGG pathway” was then used.Then, the Omics Fusion web platform ; age effect—AE (samples from the control plants at 2 and 55 DAT); short-term stress—STS (the control and the stressed plants at 2 DAT); long-term stress 1—LTS1 (control and stressed plants at 55 DAT); and long-term stress 2—LTS2 (the stressed plants at 2 and 55 DAT). Each dataset had five biological replicates per treatment.Partial least squares discriminant analysis (PLS-DA) permutation tests were performed using the leaves all treatment data set to validate the model, applying permutation number = 2,000. PLS-DA is an important and widespread classification model for untargeted metabolomics, where model tendencies are described based on group separation. Groups and samples behavior can be studied through the insights given by PLS-DA on outliers, intragroup separation tendencies, and explained variance used, which add the visualization aspect to the metabolomics model. When evaluated by group separation distance, the probability that the model was created by chance was 0.06 for the lipidic-positive fraction and <0.0005 for the other two fractions .p-value cutoff of 8.0E-5 that were submitted to functional interpretation via analysis in the MS Peaks to Pathway module. After applying the initial criteria of metabolite selection, 367 peaks with a hit to just one known compound were submitted to the pathway topology analysis module, resulting in 13 pathways with a Raw p ≤ 0.05; they were: ubiquinone and other terpenoid-quinone biosynthesis; arginine biosynthesis; lysine biosynthesis; phenylpropanoid biosynthesis; monobactam biosynthesis; steroid biosynthesis; carotenoid biosynthesis; flavone and flavonol biosynthesis; tyrosine metabolism; valine, leucine, and isoleucine biosynthesis; porphyrin and chlorophyll metabolism, and Indole alkaloid biosynthesis. These pathways came out with 19, 11, 7, 20, 6, 19, 18, 6, 8, 10, 18, and 3 differentially expressed metabolites with the highest level of significance within the set of matched metabolites submitted to analysis, respectively (data not shown).The ANOVA analysis using the leaves all treatments data set generated 4,464 peaks with a P-value (FDR) ≤ 0.05, and Log2 (FC) > 0 (up-regulated) or Log2 (FC) < 0 (down-regulated)  peaks out of the 5,376 seen in the three fractions were differentially expressed—a differentially expressed peak (DEP) is a peak with an adjusted gulated) .The short-term stress dataset from leaves was employed to evaluate how distinct are the metabolome profiles of the control and stressed plants at 2 DAT, just 1 day before the leaves started to wilt. The samples applied to evaluate the STS scenario in the leaves contained 829, 589, and 4,010 peaks, respectively, and a total of 1,550 (28.56%) peaks out of the 5,428 seen in the three fractions were differentially expressed .The LTS1 dataset from leaves was employed to evaluate how distinct are the metabolome profiles of the control and stressed plants at 55 DAT. The samples applied contained 788, 580, and 4,001 peaks, respectively, and a total of 1,285 (23.93%) peaks out of the 5,369 seen in the three fractions were differentially expressed . The LTSvia analysis in the MS Peaks to Pathway module, with a p-value cutoff of 1.0E-5. After applying the initial criteria of metabolite selection, 197 DEPs with a hit to just one known compound were submitted to the pathway topology analysis module, resulting in 16 pathways with a raw p ≤ 0.05; they were: valine, leucine, and isoleucine biosynthesis; phenylpropanoid biosynthesis; arginine biosynthesis; monobactam biosynthesis; flavone and flavonol biosynthesis; indole alkaloid biosynthesis; pyruvate metabolism; lysine biosynthesis; glycine, serine, and threonine metabolism; beta-alanine metabolism; pantothenate and CoA biosynthesis; glyoxylate and dicarboxylate metabolism; C5-branched dibasic acid metabolism; fructose and mannose metabolism, anthocyanin biosynthesis, and tyrosine metabolism  to validate the model, applying permutation number = 2,000. When evaluated by group separation distance, the probability that the model was created by chance was 0.011 (polar-positive), 0.0005 (polar-negative), and 0.0415 (lipidic-positive) .p ≤ 0.05, submitted to functional interpretation via analysis in the MS Peaks to Pathway module, with a p-value cutoff of 1.0E-5. After applying the initial criteria of metabolite selection, 326 peaks with a hit to just one known compound were submitted to the pathway topology analysis module, resulting in 10 pathways with a raw p ≤ 0.05; they were: steroid biosynthesis; lysine biosynthesis, ubiquinone, and other terpenoid-quinone biosynthesis; arginine biosynthesis; monobactam biosynthesis; carotenoid biosynthesis; valine, leucine, and isoleucine biosynthesis; tyrosine metabolism; phenylpropanoid biosynthesis; and porphyrin and chlorophyll metabolism. These pathways came out with 20, 7, 17, 10, 6, 18, 10, 8, 16, and 16 differentially expressed metabolites with the highest level of significance within the set of matched metabolites submitted to analysis, respectively (data not shown).The ANOVA analysis using the roots all in treatments dataset generated 3,600 peaks with a The age effect data set from roots was employed to evaluate how distinct are the metabolome profiles of the control plants at 2 and 55 DAT, a 53-day gap between the two assessments. The AE data set contained 636, 392, and 3,976 peaks, respectively, in the polar-positive, polar-negative, and lipidic-positive fractions; and a total of 2,588 (51.72%) peaks out of the 5,004 seen in the three fractions were differentially expressed .The STS dataset from roots was employed to evaluate how distinct are the metabolome profiles of the control and stressed plants at 2 DAT, just 1 day before the leaves started to wilt. The samples applied to evaluate the STS scenario in the leaves contained 666, 429, and 3,993 peaks, respectively, and a total of 390 (7.67%) peaks out of the 5,088 seen in the three fractions were differentially expressed .The LTS1 data set from roots was employed to evaluate how distinct are the metabolome profiles of the control and stressed plants at 55 DAT. The samples applied contained 561, 380, and 3,748 peaks, respectively, and a total of 160 (3.41%) peaks out of the 4,689 seen in the three fractions were differentially expressed . A princvia analysis in the MS Peaks to Pathway module, with a p-value cutoff of 1.0E-5. After applying the initial criteria of metabolite selection, 83 DEPs with a hit to just one known compound were submitted to the pathway topology analysis module, resulting in eight pathways with a raw p ≤ 0.05. The indicated pathways were: galactose metabolism; valine, leucine, and isoleucine biosynthesis; glucosinolate biosynthesis; glycine, serine, and threonine metabolism; pantothenate and CoA biosynthesis; arginine and proline metabolism; C5-branched dibasic acid metabolism; and biosynthesis of secondary metabolites—other antibiotics. These pathways came out with 6, 5, 9, 5, 6, 4, 5, 2, and 2 differentially expressed metabolites with the highest level of significance within the set of matched metabolites submitted to analysis, respectively (data not shown).All 390 peaks differentially expressed in the STS were submitted to functional interpretation via analysis in the MS Peaks to Pathway module, with a p-value cutoff of 1.0E-5. After applying the initial criteria of metabolite selection, 39 DEPs with a hit to just one known compound were submitted to the pathway topology analysis module, resulting in seven pathways with a raw p ≤ 0.05; they were: glyoxylate and dicarboxylate metabolism; biosynthesis of secondary metabolites—other antibiotics; beta-alanine metabolism; citrate cycle (TCA cycle); monobactam biosynthesis; alanine, aspartate, and glutamate metabolism; and pantothenate and CoA biosynthesis. These pathways came out with 5, 2, 3, 3, 2, 3, and 3 differentially expressed metabolites with the highest level of significance within the set of matched metabolites submitted to analysis, respectively (data not shown).All 160 peaks differentially expressed in the LTS1 were submitted to functional interpretation via analysis in the MS Peaks to Pathway module, with a p-value cutoff of 1.0E-5. After applying the initial criteria of metabolite selection, 262 DEPs with a hit to just one known compound were submitted to the pathway topology analysis module, resulting in 10 pathways with a raw p ≤ 0.05; they were: steroid biosynthesis; lysine biosynthesis; carotenoid biosynthesis; valine, leucine, and isoleucine biosynthesis; ubiquinone and other terpenoid-quinone biosynthesis; monobactam biosynthesis; arginine biosynthesis; phenylpropanoid biosynthesis; porphyrin and chlorophyll metabolism; and glyoxylate and dicarboxylate metabolism. These pathways came out with 20, 7, 16, 10, 14, 5, 8, 15, 14, and 9 differentially expressed metabolites with the highest level of significance within the set of matched metabolites submitted to analysis, respectively (data not shown).At last, all 3,055 peaks differentially expressed in the LTS2 were submitted to functional interpretation As the goal of this study was to look for metabolites whose behavior could give insights into the salt resistance mechanisms behind the adaptation response, both in the leaves and the roots, a series of filters were applied to select peaks differentially expressed before undergoing analysis in the MS Peaks to Pathway module of the MetaboAnalyst 5.0.* FC_LTS2 * (1/FC_AE)); the log2 was then subsequently calculated. After removing those peaks non-differentially expressed in the AE and the LTS_Final scenarios, the remaining 4,126 ones underwent analysis in the MS Peaks to Pathway module. A total of 353 differentially expressed peaks with a single matched compound were submitted to the Pathway Topology Analysis module .First, the 5,354 peaks in the leaf samples common to the AE, STS, and LTS2 scenarios were selected. After that, the combined effect of the salt in the short and long-term stress—denominated LTS_Final—was calculated using the following equation: FC_LTS_Final = .At last, there were 4,954 peaks in the root samples that were common to the AE, STS, and LTS2 scenarios. After submitting them to the same treatment mentioned above, a group of 3,557 peaks was left for analysis in the MS Peaks to Pathway module. A total of 309 differentially expressed peaks with a single matched compound were submitted to the Pathway Topology Analysis module , resultiOne of the manners by which salt affects plants is through the osmotic effect. Salt reduces the osmotic potential and, consequently, diminishes the soil water potential, making it difficult for plants to absorb water  through a series of enzymatic reactions  is one of the three metabolites from this pathway that only differentiated in the long-term stress, experiencing an increase of 3.56X in its peak intensity due exclusively to the salt effect. The other two differentiated metabolites were Sinapic acid (C00482) and N1,N5,N10-Tricaffeoyl spermidine (C18070), which experienced a decrease of 96 and 54%, respectively, also due exclusively to the salt effect.Ferulic acid (C01494) and Coniferyl aldehyde (C02666) were the two metabolites that experienced the most increase in expression level in the long-term—due exclusively to the salt effect—among those 21 metabolites differentially expressed in this pathway, experiencing 9.11X and 20.45X increase in intensity, respectively . FerulicBesides ferulic acid and coniferyl aldehyde, three other metabolites upregulated at 55 DAT in the stressed plants, in comparison with the control plants also at 55 DAT; they were: 1-O-Sinapoyl-beta-D-glucose (C01175), Cinnamaldehyde (C00903), and 4-Coumaroylshikimate (C02947), which experienced an increase of 3.32X, 1.46X, and 2.22X, respectively (data not shown). According to Chun et al. , enzymesCcoAOMT1 gene in A. thaliana and demonstrating that the mutants became phenotypically hypersensitive to salt stress, Chu and colleagues provided molecular and genetic evidence indicating the importance of enhanced lignin accumulation in the cell wall of this plant species during the responses to salt stress  and LTS2 (60.1%) ones, in the roots of gliricidia plants. The principal component analysis (PCA) did not completely separate the control and stressed samples in the polar positive and the lipidic positive fraction , and allp ≤ 0.05. However, only the biosynthesis of secondary metabolites—other antibiotics one suffered the utmost impact seen, with two lignans downregulated in the short-term and kept at a low level in the long-term stress (+)-Lariciresinol (C10646), and (–)-Lariciresinol (C20454)  . This fi(C20454) , who shoIn the roots, the steroid biosynthesis is the most affected pathway in the long-term stress, with 20 differently expressed metabolites. Fourteen of them are from the phytosterol biosynthesis module . When evaluating the changes experienced by these 20 metabolites in the short-term and long-term stress, none of them expressed differentially at 02 DAT . LathostOver 250 different plant sterols are known, among which campesterol, sitosterol, and stigmasterol are the most abundant ones in most plants  pathway, and no evidence exists for its cytosolic synthesis  occurs in plastids dapA gene in G. sepium is Lys-insensitive or that the amount of Lys is not enough to trigger a feedback inhibition -4-Hydroxy-2,3,4,5-tetrahydrodipicolinate—HTPA (C20258) metabolite experienced, by far, the utmost increase (11.47X) in expression level in the long-term after removing the age effect; and the LL-2,6-Diaminopimelic acid (C00666) the utmost decrease (94%). Since HTPA expression upregulates due to salt stress, one can infer that the protein expressed by a putative homolog of the via two salt-excluding mechanisms, starting in the canopy—severe defoliation—and concluding in the roots—limited entry of Na.In the salt-stressed gliricidia plants, the osmotic shock caused by the abrupt addition of salt to the substrate led to severe defoliation. After a metabolic adjustment to that stressful situation, those plants released new leaves and continued their development. These salt-adapted gliricidia plants only absorbed a tiny part of the Na present in the substrate while absorbing Mg, Ca, and K just like the control ones. The limited entry of Na and the maintenance of the nutritional balance in the roots, plus the fact that the roots metabolome profiles of the control and adapted plants were very similar, led us to postulate that these plants are salt-excluding plants that are adapted to high salinity stress This present study not only corroborates our previous study that indicated that the phenylpropanoid biosynthesis pathway has a role in the response of gliricidia plants to a very high level of salinity . The authors confirm that the funder had no influence over the study design, the content of the article, or the selection of this journal.The grant  for this study was awarded by the Brazilian Ministry of Science, Technology, and Innovation (MCTI) The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest.All claims expressed in this article are solely those of the authors and do not necessarily represent those of their affiliated organizations, or those of the publisher, the editors and the reviewers. Any product that may be evaluated in this article, or claim that may be made by its manufacturer, is not guaranteed or endorsed by the publisher.


Mild to moderate hearing loss is common in patients with mucopolysaccharidosis (MPS) IVA. The hearing loss can be conductive, sensorineural, or mixed. However, in these patients, the mixed form is frequent, attributed to the combination of conductive and neurosensory elements, with slowly progressive evolution. Conductive hearing loss may be secondary to recurrent upper respiratory tract infections, serous otitis media, and deformities of the ear ossicles due to the accumulation of glycosaminoglycans (GAGs). Meanwhile, the sensorineural form is mainly attributed to the accumulation of GAGs in the auditory system.The aim of this scoping review is to understand the extent and type of evidence in relation to the physiopathology, classification, epidemiology, and clinical management of hearing loss and the effect of therapy for hearing loss in patients with MPS IVA.This scoping review includes participants across all genders and of no particular age group who are diagnosed with MPS IVA and develop hearing loss as a comorbidity. No exclusion criteria  will be applicable. The information sources will include experimental and quasi-experimental, analytical observational, observational, and qualitative studies. Unpublished literature will not be covered. Grey literature will be covered. A total of 2 independent reviewers will participate in the process of screening the literature, paper selection, and data extraction, and this process will be performed blindly. When all manuscripts have been selected, disagreements that arise between the 2 reviewers at each stage of the selection process will be resolved through discussion or with an additional reviewer. Results will be reported with descriptive statistics and information will be displayed in a diagrammatic or tabular manner, as explained in the JBI guidelines.The literature search was performed in November 2021 in MEDLINE, LILACS (Literatura Latino-Americana e do Caribe em Ciências da Saúde), the Cochrane Library, ScienceDirect, Google Scholar, and OpenGrey; a total of 780 results were retrieved. Completion of the review is expected in mid-2022.This scoping review will be the first to describe the extent of the information regarding the development of hearing loss in the MPS IVA population. The data gathered by this review may lead to an understanding of the grade of hearing loss in this population and allow for the assessment of possible interventions according to the disease pattern.PRR1-10.2196/32986 Mucopolysaccharidosis (MPS) IVA, also known as Morquio syndrome, is a lysosomal storage disease caused by the loss of function of the enzyme N-acetylgalactosamine-6-sulfate sulfatase, which is required for the catabolism of glycosaminoglycans (GAGs), such as keratan sulfate and chondroitin 6-sulfate . BiochemThe life expectancy of patients with MPS IVA ranges from 8 to 43 years. Respiratory failure is the primary cause of death in patients with MPS IVA, followed by cardiac failure, posttraumatic organ failure, postoperative complications, and myocardial infarction , so therWhat is the current state of evidence on the physiopathology, classification, epidemiology, and management or treatment of hearing loss and the effect of therapy for hearing loss in patients with MPS IVA?The proposed scoping review will be conducted in accordance with the JBI methodology for scoping reviews .Studies that include participants of both genders and of no particular age who are diagnosed with MPS IVA and who developed hearing loss as a comorbidity will be selected.The following studies will be selected for this review:Studies including patient hearing loss details, such as physiopathology, classification of hearing loss , and epidemiology (data on hearing loss in patients with MPS IVA according to age and gender)Studies providing information about the effects of replacement therapy on hearing loss in patients with MPS IVAStudies providing information about the medical management of patients with hearing loss due to MPS IVA and complicationsThis scoping review will consider both experimental and quasi-experimental study designs including randomized controlled trials, nonrandomized controlled trials, before-and-after studies, and interrupted time series studies. In addition, observational studies including prospective and retrospective cohort studies, case-control studies, and analytical cross-sectional studies will be considered for inclusion. This review will also consider observational study designs, including case series, individual case reports, and descriptive cross-sectional studies for inclusion. Qualitative studies will also be considered for inclusion.The search strategy will aim to identify published studies. Unpublished literature will not be included. The search strategy will focus on published studies without discriminating based on publication date or language. The key terms used in the article titles and abstracts as well as the articles’ index terms were used to develop a full search strategy for the following databases: MEDLINE, LILACS (Literatura Latino-Americana e do Caribe em Ciências da Saúde), Web of Science, the Cochrane Library, Trip Medical Database, Embase, ScienceDirect, and Google Scholar. Furthermore, a second search will be done to identify grey literature in OpenGrey and the Grey Literature Report.To identify relevant articles, we established the following systematic search strategy using MeSH  keywords: “Hearing Disorders” OR “Hearing Loss” AND “Mucopolysaccharidosis IV” or “Hearing Disorders” OR “Hearing Loss” AND “Mucopolysaccharidosis IV”.The search strategy, including all identified keywords and index terms, will be adapted for each included database and information source. Search terms will be created by the entire team of reviewers, though the search will be conducted by 1 person. The reference list of all included sources of evidence will be screened for additional studies. Studies published in any language will be included.All identified citations will be collated and uploaded into Rayyan QCRI23 (Rayyan Systems Inc) and duplicates will be removed. Titles and abstracts will then be screened by 3 independent reviewers for the application of the selection criteria. Then, the full text of the selected studies will be assessed in detail to verify inclusion. Reasons for the exclusion of studies at this stage that do not meet the inclusion criteria will be recorded and reported in the scoping review. Any disagreements that arise between the reviewers at each stage of the selection process will be resolved through discussion or with an additional reviewer. The results of the search and the study inclusion process will be reported in full in the final scoping review and presented in a PRISMA-ScR  flowchart .A data extraction instrument was created . A totalThis will likely be a descriptive tabulation of all pertinent information regarding hearing loss in patients with MPS IVA, including physiopathology, risk factors, epidemiology, diagnosis, and prevention. Findings and recommendations, methods incorporated and their usefulness, and study limitations will also be captured. All knowledge gathered will be summarized and discussed.Snowballing or citation tracking criteria will be used to identify important articles relevant to the topic of interest; this will also be done by using the reference list of a paper or citations of a paper by other articles to identify additional manuscripts relevant to the topic of the study. After identifying potential new manuscripts and citations, a backward snowballing search will be done by reviewing the reference list and excluding papers that do not fulfill the basic criteria for inclusion. Subsequently, papers from the list that have already been examined will be removed. If a paper meets the inclusion criteria, potential new manuscripts will be identified by checking the reference list of the included paper . FollowiThe search was performed on March 2, 2022, in MEDLINE, LILACS, the Cochrane Library, ScienceDirect, Google Scholar and OpenGrey; a total of 780 results were retrieved. Completion of the review is expected in mid-2022. All potential manuscripts will be imported into the reference management software Rayyan QCRI23. The results of this scoping review will identify and describe the epidemiology, physiopathology, classification, and clinical management of hearing loss in patients with Morquio syndrome. Additionally, it will also identify the known available effect of the current therapies (enzyme replacement therapy [ERT] and hematopoietic stem cell transplantation [HSCT]) on hearing loss in patients with MPS IVA.The clinical spectrum of hearing loss in MPS IVA is wide and not well described yet. There has been limited research on the audiological assessment of patients with MPS IVA. However, some studies have suggested that the prevalence of hearing loss in this population varies between 67% to 94% . AccordiThe efficacy of ERT on the audiological disorder of MPS IVA is still unknown, though a case report described an improvement with ERT therapy . However


The Human Immunodeficiency Virus (HIV) and infections related to it can affect multiple sites in the hearing system. The use of High Activity Anti-Retroviral Therapy (HAART) can cause side effects such as ototoxicity. Thus, no consistent patterns of hearing impairment in adults with Human Immunodeficiency Virus / Acquired Immune Deficiency Syndrome have been established, and the problems that affect the hearing system of this population warrant further research.This study aimed to compare the audiological and electrophysiological data of Human Immunodeficiency Virus-positive patients with and without Acquired Immune Deficiency Syndrome, who were receiving High Activity Anti-Retroviral Therapy, to healthy individuals.It was a cross-sectional study conducted with 71 subjects (30–48 years old), divided into groups: Research Group I: 16 Human Immunodeficiency Virus-positive individuals without Acquired Immunodeficiency Syndrome ; Research Group II: 25 Human Immunodeficiency Virus-positive individuals with Acquired Immunodeficiency Syndrome ; Control Group: 30 healthy subjects. All individuals were tested by pure-tone air conduction thresholds at 0.25–8 kHz, extended high frequencies at 9–20 kHz, electrophysiological tests .Research Group I and Research Group II had higher hearing thresholds in both conventional and high frequency audiometry when compared to the control group, prolonged latency of waves I, III, V and interpeak I–V in Auditory Brainstem Response and prolonged latency of P300 Cognitive Potential. Regarding Middle Latency Responses, there was a decrease in the amplitude of the Pa wave of Research Group II compared to the Research Group I.Both groups with Human Immunodeficiency Virus had higher hearing thresholds when compared to healthy individuals  and seemed to have lower neuroelectric transmission speed along the auditory pathway in the brainstem, subcortical and cortical regions. The human immunodeficiency virus (HIV) causes Acquired Immune Deficiency Syndrome (AIDS), the devastating pandemic that continues to affect millions of people worldwide.23 HIV infection and AIDS are distinct nosological entities. Many HIV-infected individuals present a normal number of immune cells, remaining asymptomatic for long periods of time, and could not be categorized as presenting the clinical definition of AIDS. To be clinically defined as AIDS, seropositive individuals older than 13 years of age, should present a CD4+ T lymphocyte count below 350 cells per mmSince the advent of new antiretroviral drugs, there was a consistent shift in the treatment of HIV infection, providing to infected individuals a delay in the disease development and improving their clinical condition, although doubts regarding the toxic action of antiretroviral drugs on both peripheral and central auditory systems.Until the 1990s, the most common treatment performed in antiretroviral therapy was the monotherapy, that is, the use of only one drug. Due to the evolution of the treatment, the combined therapy AIDS and the therapies developed to combat it have many side effects. Specifically, auditory and vestibular disorders afflict 5–34% of adults with HIV/AIDS.The HIV and infections related to it can affect multiple sites in the hearing system causing observable abnormalities in patients including altered tympanograms, as well as threshold audiograms and auditory brainstem responses. Sensorineural hearing loss associated with HIV/AIDS may result from Central Nervous System (CNS) neoplasms, from ototoxic drug administration,Despite these factors, no consistent patterns of hearing impairment in adults with HIV/AIDS have been established.This cross-sectional, observational and descriptive study was conducted at the Laboratory for Hearing Research in Auditory Evoked Potentials of the Speech and Hearing Sciences Department of the Faculdade de Medicina da Universidade de São Paulo (FMUSP) between 2012 January and 2014 December. Research methods were approved by the Ethics Committee for Analysis of Research Projects (CAPPesq) of the Clinical Board of Hospital das Clínicas and FMUSP under protocol number 1026/04. All participants signed an informed consent form. This study had been carried out in accordance with Declaration of Helsinki.The sample consisted of 71 individuals between the ages of 30 and 48, divided into three groups. The Research Group I (RGI) included 16 HIV-positive patients without AIDS, whose status were confirmed by serology and who had never received any antiretroviral treatment. The Research Group II (RGII) included 25 HIV-positive patients with AIDS, whose status were confirmed by serology. All patients in RGII were receiving HAART (combination therapy), consisting of at least three of the following drugs: lamivudine, zidovudine, efavirenz, didanosine, nevirapine, lopinavir-r, tenofovir, stavudine, indinavir, abacavir, amprenavir, ritonavir, and atazanavir.Finally, the Control Group (CG) consisted of 30 healthy subjects with reported and confirmed HIV-negative status, no history of psychiatric and neurological disease, no hearing, language, or auditory processing complaints.Individuals in the RGI and RGII were referred to the study by the House of AIDS – Zerbini Foundation  and the City Health Services Specialized in Sexually Transmitted Diseases (STD/AIDS) of the City of São Paulo Health Department.Exclusion criteria for all three groups were: pure tone audiometry from moderately severe to profound hearing loss, pregnancy, presence of opportunistic infections in activity, history of otologic surgery or history of non-HIV-related disease, presence of any cognitive impairment that could affect hearing tests results .Data regarding HIV infection, exposure category, as well as history of use of antiretroviral and other drugs with ototoxic potential were obtained from the medical records of patients in RGI and RGII. Interviews were conducted to assess the presence of risk indicators for hearing loss. Visual inspection of the external ear canal (Heine otoscopy) was performed to identify any possible obstructions by cerumen or foreign bodies that could interfere with the hearing tests.Screening immittance measurements  were carried out with AT 235 (Interacoustic) to verify the middle ear conditions. Pure-tone air conduction thresholds at all frequencies from 0.25 to 8 kHz and in the extended high frequencies at 9, 10, 12.5, 14, 16, 18 and 20 kHz were carried out with a GSI 61 Clinical Audiometer  using standard audiometric techniques in a sound-attenuated testing room.A classification to determine the grade of hearing loss and to classify the type of hearing loss was used: conductive, sensorineural, mixed or isolated hearing loss in high frequencies.19The electrophysiological tests  were carried out in an electric- and sound-attenuated testing room. Electrophysiological evaluation was made using a two-channel electroneuromyograph . Standard Bio-logic TDH-39 phones were used to deliver the sound stimuli for the electrophysiological tests. Electrodes were placed on the forehead (Fpz), left and right mastoids (M1 and M2), and left and right temporal-parietal junctions (C3 and C4) according to the standard International Electrode System (IES). Impedance values were maintained below 5 kΩ.To ABR testing, a rate of 19 clicks per second with 0.1 μs duration was used with a filter slope of 12 dB/octave, with the high filter setup at 100 Hz and the low filter at 1500 Hz and 2000 sweeps. The stimulus was 80 dBnHL. ABR measurements were duplicated to ensure fidelity. The absolute latencies of waves I, III and V, and interpeaks I–III, III–V, I–V were analyzed.The MLR was obtained with a monaural click presented at 70 dBnHL at a rate of 9.9 clicks per second, with a 10–300 Hz band-pass filter, for a total of 1000 stimuli. Na and Pa wave latencies and Na–Pa amplitudes were obtained contra-laterally  and ipsi-laterally .Individuals were asked to remain with their eyes closed during the recording of Cognitive Potentials – P300, to control the eye-movement artifacts. The oddball paradigm was used in P300 recordings. This paradigm was based on distinguishing between a target stimulus repeated randomly (20% of the time) and the non-target stimulus with frequent repetition (80% of the time). Subjects were asked to count the target stimuli whenever they discriminated them. Monaural auditory stimulus was presented. Frequencies were 1000 Hz for the frequent stimulus (non-target) and 1500 Hz for the rare (target) stimulus. The stimulus was set at 75 dBnHL. A rate of 1.1 tone-burst per second was used with the low filter setup at 30 Hz and the high filter at 1 Hz and 300 sweeps. For the P300, non-target stimuli were subtracted from target stimuli and the latency was measured at the highest positive point (amplitude) from 250 to 650 ms.Component analysis of the electrophysiological tests was performed by the lead researcher and by a second experienced researcher in the field of electrophysiology. The electrophysiological recordings were evaluated blindly; the researchers did not know about the association participant/group.Results were sent to the care provider institutions of the individuals in RGI and RGII. In case of abnormal results, patients were referred for ENT evaluation and instructed to return for reassessment after three months.p-value of 0.05 was considered significant, and was designated with an asterisk (*).Statistical analyses were conducted with ANOVA (one factor), Tukey test and Fisher's exact test. We also calculated odds ratio and descriptive measures. Initially, the left and right ears of each group were compared for each test. As no differences were found, ears were grouped and then compared. A p = 0.218), age (p = 0.119), hearing complaints (p = 0.064), CD4+ T lymphocyte counts (p = 0.193) and time of HIV infection (p = 0.168).p = 0.064); 61% of individuals in the RGI group and 89% of the RGII had at least one complaint of hearing.The average hearing thresholds in conventional and high frequency audiometry were lower for CG than observed in the other two groups . The difThe frequent occurrence of hearing abnormalities in individuals with HIV/AIDS has been long known,Regarding hearing complaints, no significant differences were observed among RGI and RGII groups; 61% of individuals in the RGI group and 89% of the RGII had at least one complaint of hearing. Several studies reported an association between HIV infection and signs/symptoms neurotological. Individuals with AIDS often have hearing complaints, because the ENT manifestations are common at any stage of the disease, leading to specific symptoms such as hearing loss, tinnitus, dizziness and ear fullness,Sensorineural hearing loss represented the most common type in the three groups, which is in agreement with previous studies.17We also observed an increased risk for hearing loss in RGII. We found a risk ratio of 6.6 for RGII, almost double the one found for RGI (3.75). The odds ratio for the presence of hearing loss was 11 for RGII and 4.66 for RGI in comparison with the CG. These finding agree with a previous work showing that HIV-positive individuals subjected to HAART had higher odds ratio for hearing loss than healthy individuals. However, these authors did not make comparisons with HIV-positive individuals who did not receive HAART. Our results contrast with those reported previouslyThe CG had lower hearing thresholds than the other two groups, and RGI had lower thresholds than RGII with significant differences in a majority of the frequencies tested. These findings suggest the existence of mechanisms that underlie the worsening of hearing thresholds in the two groups of patients with HIV/AIDS: viral presence in the cochlea previouslyIt is worth noting that sensorineural hearing loss more often affects patients with severe HIV infection.Regarding ABR, the CG had shorter latencies than RGI and RGII, which were similar between them. Other previous studies have compared the ABR of HIV-positive patients and controls.HIV can affect the subcortical and cortical areas of the CNS, and the ABR-generating system depends on the temporal synchronization of neuronal activity. Thus, the central conduction of auditory information in patients with various degrees of HIV infection may be affected, which can generate changes to ABR waves, often causing increased latencies of waves III and V, whereas waves I and II maintain normal absolute latencies.Regarding ABR in individuals with HIV/AIDS, these results indicate the usefulness of this procedure for the detection of early signs of neurodegeneration in this population, and in monitoring how fast lesions evolve.Regarding MLR, we observed significant differences in latency and amplitude of Na and Pa waves with the electrode positioned at C3. In two other studies using MLR in HIV/AIDS patients, one found a tendency toward the increase in latency and reduction in amplitude of the PA wave,This result may be explained by the fact that the first CNS alteration in this population includes subcortical demyelination, even before clinical neurological manifestations are present.Concerning P300, we found significant differences among the three groups for the P3 wave, with shorter latencies the CG and no differences between RGI and RGII. Previous studies have used long latency auditory evoked potentials to assess the central auditory pathway of individuals with HIV/AIDS.39The novel approach of the present study was to simultaneously evaluate the peripheral and central auditory pathways of HIV-positive individuals subjected or not to HAART, and to compare them to healthy individuals. Our findings suggest that HIV-positive individuals may develop alterations of the peripheral and central auditory systems that may result from direct viral action, from the presence of opportunistic infections, and/or from the use of ototoxic drugs.Regarding the number of individuals, it is worth mentioning that some challenges were found to enlarge the sample, especially in relation to the referral of the RGI individuals, once the HIV positive population who do not underwent antiretroviral treatment is scarce. For this reason, it was not possible to achieve the same number of individuals in the RGII.Specifically concerning RGII, since each patient received at least three medications among those available for HAART, it was not possible to obtain a homogeneous sample as to the type of medication used.-Elevated hearing thresholds in behavioral hearing evaluations , where the group subjected to HAART had the highest thresholds.-Longer latencies regarding waves I, III and V as well as interpeak I–V for ABR, suggesting lower speeds of neuroelectric impulse transmission throughout the brainstem auditory pathway.-Longer P300 latency, suggesting reduced processing speed of auditory information in cortical regions.-A reduction in the amplitude of the Pa wave in MLR in the comparison of RGII with RGI, suggesting alterations to the auditory pathway in cortical and subcortical regions.HIV-positive patients with and without AIDS, who were receiving HAART, when compared to healthy individuals, presented:The authors declare no conflicts of interest.