This study aimed to determine the impact on hearing prognosis of the coherent frequency with high magnitude-squared wavelet coherence (MSWC) in video head impulse test (vHIT) among patients with sudden sensorineural hearing loss with vertigo (SSNHLV) undergoing high-dose steroid treatment. This study was a retrospective cohort study. SSNHLV patients treated at our referral center from December 2016 to December 2020 were examined. The cohort comprised 64 patients with SSNHLV undergoing high-dose steroid treatment. MSWC was measured by calculating the wavelet coherence analysis (WCA) at various frequencies from a vHIT. The hearing prognosis were analyzed using a multivariable Cox regression model and convolution neural network (CNN) of WCA. There were 64 patients with a male-to-female ratio of 1:1.67. The greater highest coherent frequency of the posterior semicircular canal (SCC) was associated with the complete recovery (CR) of hearing. After adjustment for other factors, the result remained robust . In the feature extraction with Resnet-50 and proceeding SVM in the horizontal image cropping style, the classification accuracy [STD] for , (over-sampling of CR vs. PR + NR), (extensive data extraction of CR vs. PR + NR), and (interpolation of time series of CR vs. PR + NR) were 83.6% [7.4], 92.1% [6.8], 88.9% [7.5], and 91.6% [6.4], respectively. The high coherent frequency of the posterior SCC was a significantly independent factor that was associated with good hearing prognosis in the patients who have SSNHLV. WCA may be provided with comprehensive ability in vestibulo-ocular reflex (VOR) evaluation. CNN could be utilized to classify WCA, predict treatment outcomes, and facilitate vHIT interpretation. Feature extraction in CNN with proceeding SVM and horizontal cropping style of wavelet coherence plot performed better accuracy and offered more stable model for hearing outcomes in patients with SSNHLV than pure CNN classification. Clinical and Translational Impact Statement—High coherent frequency in vHIT results in good hearing outcomes in SSNHLV and facilitates AI classification. I.Sudden sensorineural hearing loss (SSNHL) is defined as sensorineural hearing loss of 30 dB or greater over at least three consecutive frequencies occurring within 72 hours In recent years, artificial intelligence has exerted its potential to reform health care delivery in otolaryngology field II.A.(IRB: KSVGH21-CT4-04). The requirement for informed consent was waived because all identifying information was removed from the dataset before analysis.This study was approved by the Institutional Review Board of Kaohsiung Veterans General Hospital, TaiwanB.We retrospectively reviewed the electronic medical records of patients with SSNHLV as their primary treatment at a tertiary medical center in Taiwan from December 2016 to December 2020. We excluded patients with hearing loss without treatment within 14 days, pre-existing hearing loss in the contra-lesioned ear over 25 dB HL, retrocochlear pathology, other otologic diseases involving hearing loss, or anterior inferior cerebellar artery infarction on magnetic resonance imaging. After the exclusions, 64 patients were enrolled in the study.Patients with SSNHL were treated with our standard protocol. Oral prednisolone  or intravenous dexamethasone (5mg/ml b.i.d.) was given for 14 days including 3–4 days course of tapering, and salvage intratympanic dexamethasone injection (5mg/ml) was performed twice per week during 2 weeks for those whose hearing recovery did not reach complete recovery.C.Pure tone audiometry and word recognition score were performed at the first visit at our clinic and followed weekly during treatment course and 2 months after treatment. The pure tone audiometry, the highly accurate exam with simulated single-test root-mean-square error about 2.7 dB HL, was the gold standard evaluation to show the hearing threshold of the patient by dB HL The hearing recovery was defined by American Academy of Otolaryngology-Head and Neck Surgery in 2019, as CR (return to within 10 dB HL of the unaffected ear in pure tone audiometry and recovery of word recognition scores to within 5% to 10% of the unaffected ear), no recovery , and partial recovery  Vestibular laboratory data were also collected within 2 weeks, including vHIT, cervical and ocular vestibular myogenic evoked potentials (c- and o-VEMP), caloric test, and posturography.D.®  according to the protocol proposed by Halmagyi et al. ® automatically reported the head and eye velocities for 0.7s, and the onset of head rotation was always relocated at 0.1s in the timeline to make sure the complete record of the VOR impulse (0.1-0.2s in the timeline) and the following saccades (0.2-0.7s in the timeline). At least 10 times exams were repeated for each six SCCs, and the mean VOR gain and the gain asymmetry value within paired SCCs were recorded and regarded as abnormal compared with the mean and standard deviation of the group of normal healthy subjects in our hospital. The asymmetry values were counted specifically according to the planes tested as the right horizontal to the left horizontal, the left anterior to the right posterior, or the right anterior to the left posterior in the way of normalized relative gain asymmetry The vHIT was performed with ICS impulseE.The analytic Morlet wavelet transform was utilized to analyze the time series data comprising nonstationary power at diverse frequencies The wavelet scale The WCA was applied to every repeated exam for each SCCs, and the MSWC of every frequency was calculated. The coherent frequency in our study was defined as the highest frequency comprising MSWC 0.9 over entire exam (0-0.7s in the timeline) evaluated from the lowest frequency in WCA. Since the velocity of head rotation was fixed in vHIT, the coherent frequency of WCA was solely affected by the responsive eye velocities of VOR impulse and following saccades.F.The algorithm of data preprocessing was made by MATLAB R2019b  and showed in For further ANN image classification, only one power spectrum wavelet coherence plot comprising the median of the coherent frequencies among repeated exams for each SCCs was chosen. For each subject, there were three specific wavelet coherence plots representative for the horizontal, the anterior, and the posterior SCCs after the selection.The methods of augmentation across imbalanced class of CR , the time and frequency windows were processed to 0.1-0.35s, 0.1-0.2s, and 0.1-0.3s started from the exam and 4-8Hz, 0-12Hz, and 0-125Hz for horizontal, vertical, and subtotal styles for image cropping of wavelet coherence plots. The period of passive head motion and median frequency of the highest coherent frequency in each SCCs were contained in these windows to make sure adequate principal component analysis of CNN classification in all three styles of image cropping. Finally, one image per patient including all three SCCs of the ipsilesional side was tiled from the left to the right or the superior to the inferior in the sequence of the horizontal, the anterior, and the posterior SCCs according to the cropping styles.G.Transfer learning in AlexNet, ResNet-18, and VGG-19 and feature extraction in AlexNet, ResNet-50, and VGG-19 with proceeding support vector machine (SVM) were employed by using the MATLAB R2019b Deep Learning Toolbox™. The CNN input was the tiled image containing time–frequency power spectrogram of the wavelet coherence between eye and head velocities in vHIT.In this study, data were stratified before partition into five-folds to establish excellent representative of the entire sample in each fold. The repeated five-fold cross-validation method was applied for iterations of training and testing.H.All statistical analyses were performed using SPSS ver. 22 . Continuous variables were analyzed using one-way analysis of variance (ANOVA), and categorical variables were compared using Pearson’s chi-squared test or Fisher’s exact test. The independent variables were determined through multivariate analysis using the forward stepwise method. A two-sided III.Ultimately, 64 patients with SSNHLV were included in the analysis , (OS of CR vs. PR + NR), (EDE of CR vs. PR + NR), and (ITS of CR vs. PR + NR) were 83.6% 7.4], 92.1% [6.8], 88.9% [7.5], and 91.6% [6.4], respectively. , 92.1%  for (CR vs. PR + NR), (OS of CR vs. PR + NR), (EDE of CR vs. PR + NR), and (ITS of CR vs. PR + NR) were 83.6% [7.4], 92.1% [6.8], 88.9% [7.5], and 91.6% [6.4], respectively, with the feature extraction with Resnet-50 and proceeding SVM in the horizontal image cropping style. However, the best performance on accuracy [STD] of pure CNN classification was only 74.8% [11.6], 82.7% [11.1], 78.9% [13.0], and 82.8% [10.0] for (CR vs. PR + NR), (OS of CR vs. PR + NR), (EDE of CR vs. PR + NR), and (ITS of CR vs. PR + NR) with Alexnet in the horizontal image cropping style. The accuracy variability of standard deviation was much more stable in CNN feature extraction with proceeding SVM classification than pure CNN analysis. Since we used image tiling to comprise the WCAs in all SCCs, the pure CNN structure was quite sensitive to differences of pixel intensity among regional areas in nature images, which may lead to misclassifications and unstable performance of model in tiled images D.There were some limitations in the current study. First and foremost, this was a retrospective study using pretrained ANN in a single institute with limited case numbers. For balanced classes and robust feature extractions, more subjects and an ANN originally designed should be adopted in the future to obtain more accurate results of classification and more stable performance of ANN models. Second, although the WCA of vHIT data could better evaluate VOR function and predict hearing prognosis in the patients with SSNHLV, the exact pathophysiology and the hypothesis of vascular insults related to poor hearing recovery and abnormal function of the posterior SCC may need further investigations in evidence of radiology or pathology. Last but not least, for clinical application, the cut-off value of the highest coherent frequency associated with complete recovery of hearing could be evaluated in the future with greater cohort.V.The high coherent frequency was a significant independent factor that was associated with good hearing prognosis in the patients who have SSNHLV. WCA demonstrated comprehensive ability in VOR function evaluation and was more robust than time series variables in prognostic predictions. CNN could be utilized to classify WCA, predict treatment outcomes, and facilitate vHIT interpretation. Feature extraction in CNN with proceeding SVM and horizontal cropping style of wavelet coherence plot performed better accuracy and offered more stable model for hearing outcomes in patients with SSNHLV than pure CNN classification.


Chiropsalmus quadrumanus inhibits caspase-1. This extract was fractionated, and the products monitored for their inhibitory activity, until the obtention of a pure molecule, which was identified as trigonelline by mass spectrometry. Moreover, four extracts inhibit cathepsin B, and Exaiptasia diaphana was selected for subsequent fractionation and characterization, resulting in the identification of betaine as being responsible for the inhibitory action. Both molecules are already found in marine organisms, however, this is the first study showing a potent inhibitory effect on caspase-1 and cathepsin B activities. Therefore, these new prototypes can be considered for the enzyme inhibition and subsequent control of the neuroinflammation.Neuroinflammation is a condition associated with several types of dementia, such as Alzheimer’s disease (AD), mainly caused by an inflammatory response to amyloid peptides that induce microglial activation, with subsequent cytokine release. Neuronal caspase-1 from inflammasome and cathepsin B are key enzymes mediating neuroinflammation in AD, therefore, revealing new molecules to modulate these enzymes may be an interesting approach to treat neurodegenerative diseases. In this study, we searched for new caspase-1 and cathepsin B inhibitors from five species of Brazilian marine invertebrates (four cnidarians and one echinoderm). The results show that the extract of the box jellyfish  Neuroinflammation is defined as an inflammatory response that occurs in the brain or spinal cord, mediated by the production and release of cytokines, chemokines, reactive oxygen species, and secondary messengers . InflammThe release of pro-inflammatory cytokines is reported in several diseases, such as Alzheimer’s disease (AD), the main type of dementia, and the degree of neuroinflammation depends on the context, duration, and course of the primary stimulus or injury ,2. RecenInflammasomes are cytosolic protein complexes, assembled after activation by Aβ peptides from AD, whose main mechanism is the activation of the NLRP3 pathway in microglia. After binding to NOD-like membrane receptors, a central domain, NATCH, favors receptor oligomerization, allowing the interaction with a caspase recruitment domain (CARD) or a pyrin domain (PYD), recruiting pro-caspases . The infThe NLRP3 inflammasome was co-localized with amyloid plaques in patients with AD. It is seen that the Tau protein is also able to activate NLRP3 and the inflammasome formation pathway . ExperimThe production of dysfunctional and toxic proteins/peptides that are the cause of other types of dementia, such as aggregated α-synuclein from Parkinson’s disease or huntingtin from Huntington’s disease, are able to activate caspase-1 through inflammasome assembly ,10.In addition to activating inflammasome, oligomeric Aβ from AD also activates cathepsin B, and then induces the production of reactive oxygen species (ROS). Furthermore, Aβ induces the cathepsin-B-mediated activation of the NLRP3 inflammasome to initiate processing of pro-caspase-1 to caspase-1, and the subsequent conversion of pro-IL-1β to active inflammatory factor IL-1β. Thus, these two enzymes share a common cytokine release pathway .Cathepsin B are mammalian cysteinopeptidases, present in most cells and tissues. They are lysosomal enzymes that act on the intracellular degradation of proteins, but they can act extracellularly when released under certain circumstances, degrading components of the extracellular matrix .In general, cathepsins B are involved in various physiological and pathophysiological processes, such as cell cycle regulation, cancer development, autophagy, and neuroinflammation . The enzIn AD, the intracellular formation of amyloid peptides causes disruption in the membrane of lysosomes, which leads to leakage of the cathepsin B from the compartment into the cytosol. In humans with AD, the concentration of cathepsin B is increased in the cytoplasm, in contrast to its location in lysosomes, and the enzyme is redistributed in the pathology . The cytOverexpression of cathepsins B, L, and X is reported in AD . Many stIn animal models genetically modified to express the mutated APP that causes AD ), cathepsin B concentration was evaluated at the protein and gene levels, and are increased ~50% in the cortex and hippocampus compared to a control group without the mutation . When thThus, obtaining new inhibitors of such enzymes (caspase-1 and cathepsin B), which are proven to be important for the development of neuroinflammation, may be an alternative to the AD treatment.In this sense, natural products can provide new molecular entities, especially marine animals, which are poorly explored from the biochemical and pharmaceutical point of view. Therefore, the discovery of new molecules could result in new prototypes that could contribute to a reduction in neuroinflammation, and be an auxiliary treatment for neurodegenerative diseases.Chiropsalmus quadrumanus extract is the only one able to inhibit caspase-1, and four extracts  inhibit cathepsin B activity, but only E. diaphana inhibits more than the known inhibitor, F-F-FMK.Extracts from five marine invertebrates belonging to two different phyla (Cnidaria and Echinodermata), shown in C. quadrumanus and Exaiptasia diaphana) were selected to be fractionated and tested again, in order to find the active molecule to be characterized. These results are shown in the subsequent sections.The extracts with best performance for both enzymes . Thus, a new fractionation step was performed c, yieldim/z was chosen for molecule identification. By comparing to databases, trigonelline (C7H7NO2) is identified with highest score, with 202 ppm error, being identified with its neutral mass and adducts.This molecule, Cqb, was analyzed by mass spectrometry, as shown in E. diaphana extract yields 11 fractions  is found, with 2.96 ppm error.The inhibitory fraction (named Ed1) was analyzed by mass spectrometry , and theNeuroinflammation is a characteristic of several types of brain diseases, caused by the release of inflammatory mediators, . In thisA high level of activity of the active enzyme caspase-1 has already been observed in the brains of patients with AD compared to patients without the disease, a result consistent with the activation of the inflammasome ,25. AlthIn a multiple sclerosis model, VX-765 reduces inflammasome assembly in the central nervous system, preventing axon injury, with consequent improvement in neurobehavior performance .C. quadrumanus) with an inhibitory effect on such enzymes. This species is still little studied from a biochemical and bioprospecting perspectives.To the best of our knowledge, this is the first time that molecules extracted from marine invertebrates have shown caspase-1 inhibitory effects. This study demonstrates the presence of a molecule from a box jellyfish , and the pleustonic polymorphic colony Velella velella (as Velella spirans) [Hydractinia and Eirene) and scyphozoans  [Xestospongia sp. [The  glucose . This co sulcata , the seaspirans) . Trigoneea spp.) ,33, and ngia sp. .C. quadrumanus extract [Trigonelline has many physiological effects demonstrated: it is able to inhibit mice caspase-3, protect pancreatic beta cells from apoptosis, induces anti-inflammatory effect with a reduction in IL-6 and IL-1β, and acts to increase the antioxidant enzymes activity . In addi extract . Finally extract .In addition to the caspase-1 inhibitor, we also found an inhibitor molecule for cathepsin B. Cathepsin B is correlated with inflammasome assembly, and cytosolic cathepsin B, resulting from lysosomes disruption after amyloid peptide action, causes caspase-1 activation . AlthougCathepsin B can also induce oxidative stress, known in AD, which may allow the interaction of thioredoxin protein (TRX) with NLRP3, and activate it, reinforcing the interaction between the two enzymes that are the objects of studies in neuroinflammation .Studies show that cathepsin B stimulates microglia to release inflammatory mediators, playing a central role in chronic inflammation. Such mediators can induce apoptosis, causing neuronal loss and cognitive decline, in addition to releasing reactive oxygen species, which can also be toxic to neurons ,42.Inhibition of cathepsin B in microglia through RNA signaling results in a reduction in Aβ-induced toxic effects . Thus, iAnother potent and selective cathepsin B inhibitor, CA-074, when administered as a prodrug (CA-074Me) in mice genetically modified for AD, causes memory improvement and reduces Aβ(1–40) and Aβ(1–42) in the brain . HoweverCrella spinulata, show enzyme inhibition with an IC50 value of 8 μg/mL [Theonella aff. mirabilis, with an IC50 of 29.0 ng/mL [Some cathepsin B inhibitor molecules have already been identified in marine invertebrates. Shishicrellastatin A and B, two steroids isolated from the Australian marine sponge  8 μg/mL . Another.0 ng/mL .5H10NO2 and monoisotopic mass 117.07897, with 0.0098 Da difference to the found mass experimental 117.0887 Da.Here, we identified the presence of betaine as a possible inhibitor of cathepsin B by using mass spectrometry. According to PubChem, the molecular formula of betaine is CLobactis scutaria, Pocillopora damicornis, Pocillopora meandrina, Montipora capitata, Porites compressa, Porites lobata, Australopsammia aurea), jellyfish , and even for the sea anemone Exaiptasia diaphana (as Aiptasia pulchella), as playing a role in regulating cellular osmotic pressure. It is interesting to notice that all the listed species have symbiotic zooxanthellae in which betaine is also detected—the only exception is the azooxanthellate coral Australopsammia aurea (former Tubastrea aurea), corroborating the autochthonous production of betaine in cnidarian tissues [Betaine (under the forms glycine betaine or proline betaine) has already been described for various species of Cnidaria, such as corals  and one species of echinoderm (Lytechinus variegatus). Marine specimens were collected on São Sebastião Island, São Paulo, Brazil , under Brazilian Environmental Agency (ICMBio/IBAMA) license #16802-2 and #13852-1.The biological material investigated comprised four species of cnidarians  in the tropical western Atlantic, with a complex and severe venom, but little is known about its life history [The only planktonic species,  history ,53.Exaiptasia diaphana is a sea anemone  distributed worldwide in tropical and subtropical waters up to 30 m deep [Exaiptasia pallida, a widely used model system for studies on symbiotic relationships with dinoflagellates and cnidarian bleaching under climate change. Palythoa caribaeorum is an abundant encrusting colonial zoanthid  distributed in intertidal and subtidal areas along the tropical western Atlantic [Renilla reniformis , formed by polymorphic colonies inhabiting unconsolidated substrates from the intertidal zone up to mesophotic areas of the tropical western Atlantic [0 m deep . The speAtlantic , from whAtlantic . The lasAtlantic . SeveralAtlantic .Lytechinus variegatus , is another abundant species originally from the sublittoral of the tropical western Atlantic [The green sea urchin, Atlantic .g for 10 min and the supernatant was lyophilized. The remaining contents were then dissolved in ultrapure water and stored at −20 °C. For the sea urchin L. variegatus, the coelomic fluid was obtained by inserting a needle into the peristomal cavity. The liquid was centrifuged at 3000 rpm and the supernatant stored at −20 °C [After sampling, animals were washed with filtered sea water and immersed in methanol containing 0.1% acetic acid for 48 h at room temperature. The resulting extract was centrifuged at 5000× t −20 °C ,60.v/v/v), at a constant flow rate of 1.0 mL/min. The eluted content was monitored by absorbance at 214 nm and the peaks were manually collected, according to the chromatogram obtained. In the case of re-fractionation, the same equipment was used, but with a C8 column , with a constant flow of 1.0 mL/min and isocratic elution, using 0.1% trifluoroacetic acid in ultrapure water. Detection was also performed by measuring the absorbance at 214 nm.Samples were fractionated by reverse-phase high-performance liquid chromatography (RP-HPLC) in Agilent 1260 Infinity equipment . Aliquots of 80 μL were inserted into the equipment, with a Phenomenex C18 column  coupled. Elution was performed by gradient from 0 to 100% B in 30 min, with mobile phase A = 0.1% trifluoroacetic acid in ultrapure water and B = acetonitrile/Milli Q water/0.1% trifluoroacetic acid  to identify low mass compounds by means of comparison with GNPS or Vaniya/Fiehn Natural Products database, both with tolerance of up to 5 ppm for the precursor.Fractions collected after HPLC were analyzed by mass spectrometry . Aliquots of 5 μL were inserted into the equipment, without a chromatographic column. Elution was performed with 50% acetonitrile containing 0.1% formic acid. The equipment was set in positive ESI ionization mode and data were collected in a full MS scan ranging from 100 to 1000 ®, Promega, Madison, WI, USA). All fluorescence values were subtracted from the background (buffer).Assays on caspase-1 were conducted in 384-well plates, in triplicate, with a final reaction volume of 20 μL using the Caspase-1 Inhibitor Screening Assay Kit . Samples (10 μg) were previously incubated with enzymes (recombinant human caspase-1) treated with 2 mM DTT for 10 min at room temperature. Buffer was added to the wells and then the synthetic substrate . A known caspase-1 inhibitor, Z-VEID-CHO , was used as a positive control. The increase in fluorescence after incubation was measured by a fluorimeter at the excitation wavelength of 400 nm and emission wavelength of 505 nm . Samples (10 μg) were previously incubated with enzymes (recombinant human cathepsin B) treated with 2 mM DTT for 10 min at room temperature. Buffer was added and then 10 mM of synthetic substrate (Ac-RR-AFC). A known inhibitor, FFFMK (10 μM), was used as a positive control. The increase in fluorescence 50 min after incubation was measured by a fluorimeter at the excitation wavelength of 400 nm and emission wavelength of 505 nm . VelocitC. quadrumanus, and betaine for cathepsin B, from the sea anemone E. diaphana. These molecules represent alternatives for the control of neuroinflammation, and may become important therapeutic targets, and may contribute to the treatment of neurodegenerative diseasesWe obtained two molecules with enzymatic inhibitory activity, viz. trigonelline for caspase-1, from the box jellyfish


TCOF1, responsible for a syndromic form of congenital hearing loss–in which deleterious variants are also associated with adult-onset hearing loss. We also identify four additional novel candidate genes  in which rare variant burden may be associated with hearing loss. Our results confirm that rare variants in Mendelian hearing loss genes contribute to polygenic risk of hearing loss, and emphasize the utility of healthcare system cohorts to study common complex traits and diseases.Loss or absence of hearing is common at both extremes of human lifespan, in the forms of congenital deafness and age-related hearing loss. While these are often studied separately, there is increasing evidence that their genetic basis is at least partially overlapping. In particular, both common and rare variants in genes associated with monogenic forms of hearing loss also contribute to the more polygenic basis of age-related hearing loss. Here, we directly test this model in the Penn Medicine BioBank–a healthcare system cohort of around 40,000 individuals with linked genetic and electronic health record data. We show that increased burden of predicted deleterious variants in Mendelian hearing loss genes is associated with increased risk and severity of adult-onset hearing loss. As a specific example, we identify one gene– Age-related hearing loss is relatively common and has a genetic component to risk, though little is known about specific genes that are involved. Here, we study participants in the Penn Medicine BioBank to identify factors that increase risk of hearing loss. We show that loss-of-function variants in genes that are known to cause Mendelian forms of hearing loss also increase risk of age-related hearing loss. Because many such genes have been identified, they can be used to interpret the results of genome-wide association studies and to investigate the biological basis of age-related hearing loss. Our results also emphasize that many reported Mendelian hearing loss variants are incompletely penetrant and may act cumulatively. Finally, our study shows how hospital-recruited biobank cohorts can aid in the study of common conditions like hearing loss, even when those conditions are not the primary reason for contact with the health system. Hearing loss (HL) is one of the most common age-related conditions. Around 50% of American adults experience some difficulty hearing and 50% of those aged 70 and over have hearing loss to a level that is disabling . EstimatUnlike children, adult patients are rarely assessed for genetic etiology when presenting with hearing loss. As current clinical management of adult-onset HL would not be significantly impacted by results of genetic testing, such investigation would have low immediate clinical utility. However, understanding the genetic basis of age-related HL may have longer-term benefits for translational therapy, particularly if such therapy can be linked to congenital HL genes, many of which are well-studied in mouse or other models. Greater understanding of HL genetics would also enable screening of high risk individuals at an earlier age who might benefit from preventive or restorative treatment options. Because age-related HL is common, many participants in hospital or healthcare system biobank cohorts are likely to have HL  providing an opportunity to study the genetic basis of age-related HL in large cohorts without specific recruitment. In this study, we use the Penn Medicine BioBank (PMBB) to investigate the extent to which deleterious variants in known congenital HL genes contribute to polygenic risk of age-related HL and attempt to identify specific genes that contribute to the phenotype .PMBB is a database of linked electronic health records (EHR), biospecimens and genetic data of participants recruited through the Penn Medicine health system . The datWe first categorized cases and controls following a common scheme in EHR studies. Phenotypes are defined in terms of phecodes–a standardized hierarchical classification of diseases and traits ,19. If a2 and 20 genomic principal components as covariates, and find that degree HL is associated with burden count . Next, we estimated the effect of burden for each degree HL independently, confirming that gene burden is associated with both the presence and severity of hearing loss (−12 for association between loss-of-function variant burden in known HL genes and HL). Though absolute effect sizes are not directly comparable between UKB and PMBB because of the different phenotypes, we noted that in UKB, as in PMBB, the effect of each ClinVar variant was four times the size of each non-ClinVar variant, the effects of variants in DFNA and DFNB genes were similar, and the effects of variants in genes that can act as both DFNA and DFNB were larger  < 0.05  , a nuclear receptor associated with autosomal recessive hearing loss [We tested for association between predicted deleterious variant burden of individual HL genes, and degree HL, using all individuals . We foun.2×10−6) , Table 3ing loss . HoweverTCOF1 cause Treacher Collins Syndrome, which involves craniofacial deformities and conductive hearing loss due to abnormal neural crest cell development [TCOF1 haploinsufficiency. We identified 8 carriers of predicted loss-of-function or pathogenic missense variants in our dataset  were significant at FDR < 0.05  gene encodes an integral protein of the inner mitochondrial membrane. Mice with Nnt mutations exhibit impaired insulin secretion, which is also known to increase risk of hearing loss [Of these 373 genes, four  of the randomized and observed results, and increased the allele frequency cutoff until inflation was not observed in the randomized results (genomic inflation constant λ = 1.01). We identified a single genome-wide significant locus upstream of ir cells . Mouse klt in HL , althougR2 –the difference in R2 between a linear model of degree HL with and without the PRS . The incremental R2 of the PRS is 0.19% (bootstrap standard error 0.054%), rising to 1% if we restrict to individuals with audiogram data , similar to individuals in the top 1% of the PRS distribution (odds ratio 2.6).Finally, we computed polygenic risk scores (PRS) using PRS-CS  and a prram data . As obseram data , and alsOur study illustrates both the opportunities and challenges of using EHR data to study a common complex condition like hearing loss. In particular, the subset of individuals in our data with audiograms makes it clear that EHR-derived phenotypes are highly inaccurate for this phenotype, perhaps because phecodes are largely constructed using hospital billing codes and hearing loss is not typically the primary reason for hospital visits. Published large GWAS studies have relied largely on self-reported hearing loss in UK Biobank ,5 which TCOF1 variants with adult-onset HL. While TCOF1 is known to cause congenital HL as part of Treacher-Collins syndrome, it has not previously been reported to increase risk of adult-onset HL. While hearing loss in Treacher-Collins syndrome is generally conductive due to malformation of middle ear structures, in at least one of our TCOF1 cases hearing loss is sensorineural, suggesting a different mechanism. This result is an example of how knowledge about genes associated with early-onset or developmental conditions can be used to prioritize gene discovery for related adult-onset conditions. Similar observations have been made about the excess contribution of genes that are essential or involved in developmental disorders to psychiatric traits such as autism [We identified and replicated a novel association of s autism  and schis autism .Our observation that rare variant burden in known HL genes is associated with both risk and severity of adult onset HL supports previous observations that common variant heritability is enriched around known HL genes  and thatWe also identified five novel genes potentially associated with HL–four through gene burden analysis and one through GWAS. Although these genes are all biologically plausible, these associations are only modestly supported and none of them replicated in the Praveen et al. study. We therefore consider these associations as only candidates until replicated. One limitation is that Praveen et al. used self-reported hearing loss as opposed to audiometric data. Another is that for rare variants we only looked at coding variation and not regulatory variation at these genes, which might explain a larger proportion of heritability.Another opportunity and challenge of our dataset is the level of ancestral diversity. Approximately 25% of the individuals in the Penn Medicine BioBank are of African American ancestry. Performing genetic discovery within and across cohorts of diverse ancestry is both a key goal and largely open question in human genetic research today ,40. WhilIn summary, we used biobank data and a prioritized list of known HL genes to identify novel associations with adult-onset HL. We showed that burden of deleterious variants in known HL genes is robustly associated with HL and identified five additional candidate genes through exome- and genome-wide analysis. Because hearing loss is so common in older individuals, healthcare cohorts like the Penn Medicine Biobank have great potential to allow gene discovery, and validation of predictive tests, even if not specifically collected for that purpose, and we expect that these approaches will become increasingly common as such cohorts expand in size.The collection, storage and analysis of biospecimens, genetic data and data derived from electronic health records as part of the Penn Medicine BioBank is approved under University of Pennsylvania IRB protocol #813913. Each participant provided written informed consent.The Penn Medicine BioBank (PMBB) has recruited approximately 60,000 participants and hosts data from patients of clinical practice sites of the University of Pennsylvania Health System. Each participant provided informed consent regarding storage of biological specimens, genetic sequencing, and access to all available EHR data. This study was approved by the Institutional Review Board of the University of Pennsylvania.rd degree. A total of 9,356 individuals considered African American ancestry and 27,151 individuals considered European ancestry were included after quality control measures including removal of individuals with missing covariates.Exome sequences were generated by the Regeneron Genetics Center . Sequencing was performed in 2020 with the custom IDT xGen v1 exome capture platform and sequenced on an Illumina NovaSeq 6000 system using S4 flow cells. Sequences were mapped and variants were annotated using ANNOVAR, gnomAD, and REVEL (Rare Exome Variant Ensemble Learner), and samples with low exome sequencing coverage (less than 85% of targeted sites at 20X coverage), apparent contamination (D-statistic > 0.4), discordance with reported sex, or that were duplicates, were removed, as previously described ,41. AfteWe extracted audiometric data from a clinical database (AudBase) of audiograms performed at the Hospital of the University of Pennsylvania audiology practice between May 5, 2013 and February 25, 2021–1,917 audiograms were available for the 36,507 study individuals. From the clinical database, we calculated pure-tone average (PTA) for bone and air conduction using the arithmetic mean of the hearing threshold in decibels at 500, 1000, and 2000 Hertz. We recorded air conduction PTA of the worse ear and assigned HL levels according to previously published categorization: PTA 0–15 (degree HL 0), PTA 16–25 , PTA 26–40 , PTA 41–55 , and PTA 56+  . To iden2, sex, and genome-wide PCs 1–20 as covariates.To select variants to include in gene burden analysis, we used a combination of minor allele frequency filters and predicted pathogenicity. First, we removed variants if their gnomAD allele frequency in African and Non-Finnish European individuals was greater than .001. We then filtered remaining variants on an allele frequency of .01 in the entire dataset. Next, we restricted to predicted loss-of-function (pLoF) variants, namely frameshift mutations, stop-gains, and those that disrupt canonical splice sites. We also included nonsynonymous missense variants after filtering to restrict to those with high predicted pathogenicity, defined as the Rare Exome Variant Ensemble Learner (REVEL) score being greater than 0.6 . We anno10(p) from the association tests without additional filtering. Briefly, we randomly permuted the phenotype and re-ran the association tests. For known HL genes, we assumed that variants in these genes would be deleterious, as deleterious variants in these genes were how they were discovered and thus we filtered based on case carriers >0 instead of thresholding based on carriers only. When expanding analyses to all genes, we again used a case carrier threshold, but were much more conservative as we were attempting novel discovery–we increased the case carrier threshold in increments of 5 until we observed very minimal test statistic inflation in the randomly permuted -log10(p) QQ-plot, and chose a threshold of >25 case carriers. To correct for multiple comparisons, we treated burden in known and novel HL genes separately, and used Benjamini-Hochberg correction.We performed permutation analyses to determine the (case) carrier cut-off and minor allele frequency thresholds for the burden tests, as we observed test statistic inflation in the QQ-plots of the -loghttps://pmbb.med.upenn.edu/data-access/data-release/2020_2_0/imputed/), and were imputed to the TOPMed reference panel [R2 >.30 and used the same modeling setup and covariates as with the burden tests. We performed association tests using Plink 1.9 [Array genotypes were processed according to these specifications  and PRS from the R2 when regressing HL just on the covariates.We calculated PRS for PMBB individuals with PRS-CS  using de2, sex, and the top 5 genetic principal components provided by UKB.We replicated the association between gene burden and HL in UK Biobank. Hearing loss cases were defined as those who answered ‘Yes’ for both “Hearing difficulty/problems” and “Hearing difficulty/problems with background noise”, and controls were individuals who answered ‘No’ for both–remaining individuals were removed. We note that this phenotyping definition is similar to previous analyses of hearing loss in UKB . 148,970We replicated novel significant gene and GWAS associations using results from Praveen et al. . BrieflyS1 Table(XLSX)Click here for additional data file.S2 Table(XLSX)Click here for additional data file.S3 Table(XLSX)Click here for additional data file.S4 Table(XLSX)Click here for additional data file.S5 Table(XLSX)Click here for additional data file.S6 Table(XLSX)Click here for additional data file.S7 Table.(XLSX)Click here for additional data file.S8 Table.(XLSX)Click here for additional data file.S1 Note(PDF)Click here for additional data file.


Although the two-component system is known to play considerable roles in sensing and responding to environmental signals, there is little information regarding the two-component systems of thermophilic cyanobacteria. Herein, we investigated the structure and architecture of two-component systems in 17 well-described thermophilic cyanobacteria. The results revealed a fascinating complexity and diversity of these systems. Moreover, the distinct composition of genes related to these systems existed among these thermophilic cyanobacteria. In addition, we found diversified domain architectures of histidine kinases and response regulators, putatively in association with various functions. Furthermore, horizontal gene transfer, as well as duplications events, might be involved in the evolutionary history of genes relevant to these systems in certain genera. The obtained data will highlight that the genomes of thermophilic cyanobacteria have a broad potential for acclimations to environmental fluctuations.Leptodesmis, Leptolyngbya, Leptothermofonsia, Thermoleptolyngbya, Thermostichus, and Thermosynechococcus. The results revealed a fascinating complexity and diversity of the TCSs. A distinct composition of TCS genes existed among these thermophilic cyanobacteria. A majority of TCS genes were classified as orphan, followed by the paired and complex cluster. A high proportion of histidine kinases (HKs) were predicted to be cytosolic subcellular localizations. Further analyses suggested diversified domain architectures of HK and response regulators (RRs), putatively in association with various functions. Comparative and evolutionary genomic analyses indicated that the horizontal gene transfer, as well as duplications events, might be involved in the evolutionary history of TCS genes in Thermostichus and Thermosynechococcus strains. A comparative analysis between thermophilic and mesophilic cyanobacteria indicated that one HK cluster and one RR cluster were uniquely shared by all the thermophilic cyanobacteria studied, while two HK clusters and one RR cluster were common to all the filamentous thermophilic cyanobacteria. These results suggested that these thermophile-unique clusters may be related to thermal characters and morphology. Collectively, this study shed light on the TCSs of thermophilic cyanobacteria, which may confer the necessary regulatory flexibility; these findings highlight that the genomes of thermophilic cyanobacteria have a broad potential for acclimations to environmental fluctuations.Two-component systems (TCSs) play crucial roles in sensing and responding to environmental signals, facilitating the acclimation of cyanobacteria to hostile niches. To date, there is limited information on the TCSs of thermophilic cyanobacteria. Here, genome-based approaches were used to gain insights into the structure and architecture of the TCS in 17 well-described thermophilic cyanobacteria, namely strains from the genus  They show a cosmopolitan distribution in most terrestrial, freshwater, and marine habitats, and even in extreme environments, from Antarctica to hot springs ,2,3. Cyamissions . HoweverAs in most prokaryotes, cyanobacteria utilize two-component systems (TCSs) as some of the major pathways to sense and coordinate their behavior in response to changes in the external environment ,7,8. TCSThe increasing number of prokaryotes with complete genome sequences has dramatically extended our cognition of TCS prevalence among prokaryotes . MoreoveIt has been reported that the utilization of the TCS in the construction of synthetic genetic networks may be useful for engineering novel cell functions, such as biofuel-related metabolic engineering in microorganisms by the implementation of genetic biosensors . In addiIn the current work, we analyzed the TCS repertoire of thermophilic cyanobacteria. The distribution and genetic/domain organization of the TCS genes were assessed. Insights were further gained for the structure and architecture of HKs and RRs. Moreover, the conserved TCS genes were identified, and comparative and evolutionary genomic analysis revealed the importance of accessory TCS genes in the expansion of TCS genes through horizontal gene transfer (HGT) and gene duplication events. In addition, we hypothesized that a unique TCS may be contained by thermophiles due to their thermal characters.Leptodesmis sichuanensis A121 [Leptolyngbya sp. JSC-1 [Leptothermofonsia sichuanensis E412 [Thermoleptolyngbya sp. O-77 [T. sichuanensis A183 [Thermostichus sp. 60AY4M2, 63AY4M2, 65AY6A5, 65AY6Li [Thermosynechococcus lividus PCC 6715 [Thermosynechococcus sp. CL-1 [T. vestitus BP-1 [T. vulcanus NIES-2134 [A dataset of 17 thermophilic cyanobacteria was retrieved from the genomic resources of the NCBI. The collection of these strains was established according to our previous study through performing a filtration based on NCBI genomic resources, validation of thermophilic characteristics, and quality control of genomic sequences . Detailesis A121 , Leptolyp. JSC-1  and Leptsis E412 ; Oculatesp. O-77  and T. ssis A183 ; Synecho 65AY6Li , JA-2-3B 65AY6Li ; ThermosPCC 6715 , Thermossp. CL-1  and TA-1sp. CL-1 , T. vesttus BP-1  and E542tus BP-1 , T. vulchttp://www.p2rp.org/, accessed on 15 May 2022) [The TCS genes in each genome were identified using P2RP (ay 2022) . All TCSay 2022) , while ‘According to the genetic organization on the DNA, TCS genes were classified as orphan when no TCS gene was adjacent to another TCS gene, as paired when two TCS genes were adjacent, and as complex when more than two genes were contiguous . Given fhttp://pfam-legacy.xfam.org/, accessed on 20 June 2022). Protein alignments were performed with the default parameters using Muscle implemented in Mega7 [The protein sequences of TCS genes were downloaded from results calculated by P2RP. Domains within protein sequences of TCS genes were identified by the PFAM tool of European Bioinformatics Institute (EMBL-EBI)  criterion, orthologous proteins of TCS genes in focal taxa were identified using BLASTP. The BLASTP alignments were performed with the following thresholds: identity percentage greater than 40% and query coverage greater than 75%.Maximum-likelihood (ML) phylogenetic analyses of HKs and RRs were carried out using PhyML v3.0 , and theThermosynechococcus NIES-2134) to 239 (Leptolyngbya JSC-1). Intergenic variations were evident in TCS gene number  of the other Thermosynechococcus strains. The result indicated that one HK might phosphorylate more than one RR in these thermophilic cyanobacteria. On the contrary, the Thermoleptolyngbya strains demonstrated a receiver/transmitter ratio of 0.9, suggesting that one RR may be phosphorylated by more than one HK. Inconsistent receiver/transmitter ratios were also observed in other cyanobacterial genomes , as suggested by the Pearson analysis.Intriguingly, the TCS gene number appeared to be positively associated with genome size . To veriLeptolyngbya sp. JSC-1 was not a phylogenetic member of the genus Leptolyngbya sensu stricto but represented a new genus within the family of Leptolyngbyaceae [Leptolyngbya sp. is not a valid name for this strain.Additionally, there is one thing to bear in mind. Previous studies strongly suggested that gbyaceae ,47. NeveThermosynechococcus genomes. Among these complexes, the number and loci organization varied among the thermophilic strains (Leptothermofonsia genome (RR-RR-cHK-RR) and Pentad in the genomes of Leptolyngbya (RR-cHK-RR-RR-hyHK), Leptothermofonsia (hyHK-RR-hyHK-hyHK-RR) and Thermoleptolyngbya (RR-RR-hyHK-hyHK-hyHK). Nevertheless, the putative phosphotransfer routes deduced from the configurations of these complexes were difficult to comprehend and will require further experimental verification.From the perspective of function, genes inside the same operon often function in the same pathway. Thus, thoroughly understanding the genetic organization of TCS genes is crucial information so as to provide a potential TCS partnership. Herein, we investigated the genetic architecture of TCS genes in genomes of the 17 thermophilic cyanobacteria . A major strains . Most ofEscherichia coli were primarily organized as pairs (71.5%), followed by the orphaned (14.5%) and complex (14.5%) [In contrast to the genetic organization of TCS genes in thermophilic cyanobacteria, the TCS genes in  (14.5%) . By compInterestingly, apart from the conventional genetic organization of TCS genes (orphan or paired), the TCS complexes tend to be enriched in cyanobacteria with larger genomes . This obThermostichus (46.7–58.8%) and Thermosynechococcus (42.9% to 72.7%). The proportion of the soluble HKs observed in thermophilic cyanobacteria was comparable to that of other cyanobacteria according to the TCS database , e.g., Nostoc punctiforme PCC 73102 (65%) and Prochlorococcus marinus str. MIT 9313 (40%). Initially, soluble HKs were considered to sense intracellular signals , supervising the internal metabolic status of bacteria [Leptodesmis, Leptolyngbya, Leptothermofonsia, Thermoleptolyngbya and Thermosynechococcus PCC6715. Such a prevalence of cytosolic hyHKs may provide a selective advantage for an organism through facilitating spatial proximity with the corresponding cognate RR, consequently improving the velocity and efficiency of the signaling route [Herein, we specifically focused on TCS proteins that were not a part of Che-like or unorthodox systems. Thus, the HK afterwards referred to cHK and hyHK. The existence of transmembrane helices was used to evaluate the cellular localization of each HK identified. In the 17 thermophilic cyanobacteria, 42.9% to 72.7% of HKs were predicted to comprise no transmembrane segment , indicatbacteria . Notwithng route .Sensing domains are responsible for sensing environmental signals and are important to comprehend the essence of the signals perceived by individual HK . In the Thermosynechococcus strains, which followed the pattern: GAF > PAS > HAMP domain. In addition, only in Leptolyngbya JSC-1 PAS domains appeared to be more abundant in hyHKs than in cHKs (61 versus 31), whereas the opposite trend was observed in the other 16 strains. PAS domains mainly function as monitoring changes in extracellular or intracellular environments by sensing chemical or physical signals, e.g., small molecules, light, redox state, ions and gases [The most abundant sensing-domain within HKs was the PAS (Per-Arnt-Sim) domain, followed by the GAF  domain and HAMP  domain . Howevernd gases . The prend gases . The HAMnd gases , though Leptodesmis, Leptolyngbya, Leptothermofonsia and Thermostichus (except for strain JA-2-3Ba). All the PHY domains predicted were either standalone PHY (Leptothermofonsia) or in a cluster with other domains in the N-terminal of cHKs, forming different types of domain arrangements, namely PAS-GAF-PHY (Leptodesmis), GAF-GAF-PHY  and GAF-PHY-PAS  (slr0473) from Synechocystis PCC 6803 [Leptolyngbya JSC-1 and Leptothermofonsia E412 has been experimentally verified [Thermosynechococcus strains, since they were typical cyanobacteria showing a blue/green-type reversible photoconversion. This should be ascribed to the cyanobacteriochromes, a group of photoreceptors distantly related to the phytochrome family [Thermosynechococcus strains.At least one photoreceptor PHY (phytochromes-specific) domain was found only in ofonsia) a. Phytocofonsia) b. A furtofonsia) c. ComparPCC 6803 , canonicPCC 6803  and the verified ,59. In ae family . Only the family . Althouge family . LikewisLeptolyngbya, Thermosynechococcus and Leptolyngbya. NIT (Nitrate- and nitrite- sensing) was only associated with Leptothermofonsia and Thermoleptolyngbya, suggesting that they may monitor the nitrate or nitrite concentration [Leptodesmis and Leptolyngbya exhibited a Ser/Thr kinase catalytic domain (S_TKc), indicating that the two cyanobacteria may also have the eukaryotic-type signal transduction systems in addition to the prokaryotic two-component systems [The remaining sensing domains showed an irregular occurrence, and some sensing domains were found to be specific to a few strains. For example, the cNMP binding, FHA and Pkinase domains were found exclusively in ntration . Leptode systems . Taken tLeptolyngbya JSC-1 demonstrated a median percentage (57.3%), which could be ascribed to the abundant hyHK domain architectures.Domain architecture is very informative for predicting the putative function of signaling proteins . In the Thermostichus and Thermosynechococcus genomes, the subsequent analysis focused on the hyHKs in Leptodesmis, Leptolyngbya, Leptothermofonsia and Thermoleptolyngbya. The hyHKs consisted of only one transmitter and one receiver domain was dominant among the hyHK domain architectures in the five genomes (71.4–77.6%), whereas the remaining hyHks showed atypical domain architectures with multiple transmitter and/or receiver domains. Those sophisticated domain architectures suggested that each protein may possess complex phosphotransfer pathways. Meanwhile, 24.5% to 50.0% of hyHKs in the five genomes contained no sensing domains, while the other hyHKs possessed multiple sensing domains: as many as 13 sensing domains  (The top 10 abundant domain architectures are summarized in a JSC-1) . These pN. punctiforme PCC 73102  [E. coli, but their roles are far beyond as they are also able to act as connectors between TCS partners, thus facilitating crosstalk, feedback, and phosphorelays within the phosphorylation network of TCS [HKs can be easily identified due to the possession of a characterized transmitter domain, whereas RRs were defined by the possession of receiver domains . Howeverk of TCS . As for Thermostichus or Thermosynechococcus strains were summarized in Thermostichus genomes, 222 (81.6%) out of 272 putative TCS genes were common to all genomes, revealing a strong conservation of TCS genes in Thermostichus strains. The core set of Thermostichus TCS genes included 72 cHKs and 150 RRs  out of 242 putative TCS genes were common to all genomes, including 48 cHKs, 12 hyHKs and 114 RRs  were acquired only by JA-2-3Ba, suggesting that the two genes may have been recently acquired by this strain; the other two HKs (JA-2-3Ba _ABD03674 and 65AY6A5_PIK87857) might be independently acquired by the two strains; and the three RRs  might be acquired by the common ancestor of the three strains. All the events that occurred in JA-2-3Ba might contribute to the divergent genomes-based clustering between this strain and other Thermostichus strains (Compared to the core set of TCS genes, accessory TCS genes might contribute more to the genome plasticity. Herein, comparative and evolutionary genomic analyses were performed to reveal the origin of accessory TCS in CS genes a,b. More strains a,b.Thermosynechococcus strains, 42 gene loss events and 26 acquisition events were putatively detected (Thermosynechococcus strains (For the 68 accessory TCS genes in the six detected c,d. A to strains c,d. More strains , indicatThermostichus and Thermosynechococcus strains. Specifically, our results were consistent with the previous report that numerous putative genes horizontally transferred from other bacteria have been actively acquired by Thermosynechococcus species, enabling the acclimation of them to stressful niches in hot springs [In summary, HGT, as well as duplication events, might be involved in the evolutionary history of TCS genes in  springs . Taken tSynechococcus PCC 7942 and Synechocystis PCC 6803 were used as references to elucidate the phylogenetic relationship of TCS between thermophilic and mesophilic cyanobacteria. As shown in Thermosynechococcus PCC 6715 was missing in the shared HK cluster of thermophiles. The further BLASTP and domain analysis suggested that after the removal of one of the two fhlA domains, a pseudogene in the genome of PCC 6715 became highly homologous to the HKs of the shared cluster. The causes of the two duplicated fhlA domains in the pseudo gene are unknown. In addition, two HK clusters and one RR cluster were only common to all of the filamentous thermophilic cyanobacteria, suggesting that these HKs and RRs may be functionally related to the morphology. This could be indicative of the putative thermosensing function of histidine kinases present in this thermophile-specific clade [Typical, well-described, mesophilic cyanobacteria ic clade . The theThermostichus strains (Thermosynechococcus PCC 6715, all the thermophilic cyanobacteria contained homologs of Rre1 (The TCS in PCC 7942, Hik2 and Rre1, was reported to be highly conserved among cyanobacteria and to be a multi-stress responsive signal-transducing module that controls the expression of hspA, a chloroplast gene regarding redox regulation and other genes in cyanobacteria ,70. Howe strains , showing of Rre1 , exhibit of Rre1 , suggest of Rre1 . This syThermostichus and Thermosynechococcus strains. A comparative analysis between thermophilic and mesophilic cyanobacteria indicated that one HK cluster and one RR cluster were uniquely shared by all the thermophilic cyanobacteria studied, while two HK clusters and one RR cluster were only common to all the filamentous thermophilic cyanobacteria. These results suggested that these thermophile-unique clusters may be related to thermal characters and the morphology. Overall, these findings provided insights into the TCS of thermophilic cyanobacteria and fundamental knowledge for further research regarding thermophilic cyanobacteria with a broad potential for acclimations to environmental fluctuations.In the current study, we conducted a comprehensive comparative analysis of the components and structures of TCS in 17 thermophilic cyanobacteria. The results revealed a fascinating complexity and diversity of the TCSs. A distinct composition of the TCS genes existed among these thermophilic cyanobacteria. The TCS genes were mainly organized as orphans, and a high proportion of HKs were predicted to be soluble. The domain architectures of HK and RRs were diversified among these thermophilic cyanobacteria, suggesting their various roles in biological functions. Comparative and evolutionary genomic analyses indicated that HGT, as well as duplications events, might be involved in the evolutionary history of TCS genes in


Aim: The goal of our study was to characterize vestibular test results from rural workers occupationally and environmentally exposed to organophosphates used in agricultural pesticides. Materials and Methods: We carried out a cohort cross-sectional study with 18 rural workers from Teresópolis, RJ. Age ranged between 16 and 59 years, with a mean value of 39.6 years, 5 were males and 13 females. We used three questionnaires with questions associated with general and auditory health, and more specific questions about dizziness and its association with work. All workers underwent clinical evaluation, audiometry and vectoelectronystagmography. Results: results showed that 16 workers had irritative peripheral body balance disorder and 7 workers had sensorineural hearing loss, thus suggesting that agricultural pesticides cause vestibular alterations through a slow and silent intoxication. Conclusions: medical care for this population exposed to neurotoxic substances require an interdisciplinary approach, to guarantee efficient preventive and therapeutic measures.Occupational and environmental exposure to agricultural pesticides represent an important health care problem in our country. Among the symptoms presented, dizziness stands out, because of a probable toxic action.  Some chemicals have caused a lot of damage to human beings and the environment. Deterioration of forests, rivers, the air and impacts on human health have been observed as a result contact with such substances, either acute or gradual.The increased presence of toxic substances has produced severe impact on the environment and the population exposed to their noxious effects. The use of pesticides, mainly in developing countries, is increasing every day. In Brazil, 5,570 cases of poisoning in an occupational context were reported in 2003, 1,748 (31.4%) by pesticide and 640 (11.5%) by other chemical products Exposure will often times produce silent clinical alterations that cannot be detected by biologic markers and that affect the lives of workers. Apart from the clinical effects observed in neurologic examination, subclinical symptoms can be assessed by means of neurophysiologic and neuropsychological testsThe US Environmental Protection Agency  defines pesticides as any substance or mixture of substances that aims to prevent, destroy, kill, or repel pests. Among the most broadly used in agriculture are anticholinesterase agents such as organophosphorous compounds that inhibit acetylcholinesterase (AChE), an enzyme that destroys the biologic activity of neurotransmitter acetylcholine (ACh). One it accumulates in the nerve ends (ACh), nerve electric stimulation does not stop. Toxicity signs include stimulation of muscarinic receptors of the parasympathetic system in the autonomous nervous systemOtotoxicity must be seen and studied as a matter of public health. Over 200 substances have been described in the literature as ototoxic, and most of them are not seen as noxious to menFor industrial toxicologists, ototoxic substances include all physical and chemical elements capable of damaging hearing. JergerHearing loss may set in rapidly or more slowly, and severity depends on the amount, time of exposure, and interaction sustained with the ototoxic agent. Hearing loss may start during exposure or even months later, and is irreversible. It is mostly bilateral and symmetric, although cases of unilateral and asymmetric hearing loss have been reported. Vestibular involvement, although often concurrent, may appear earlier and more intensely than hearing symptoms.Many papers indicate that dizziness is a subjective symptom related to pesticide exposure.Martin, Adams (2003)7 stress the importance of health care workers being aware of the symptoms connected to the use of chemical and biological weapons, so that quick and accurate diagnosis can be produced. Organophosphorous compounds, for instance, were characterized by their neurologic impacts and unspecific symptoms such as headache and dizziness.Teixeira et al. (2003)Delgado, Paumgartten (2004)9 interviewed 55 farmers on the use of pesticides through a questionnaire. They observed that 92% of the workers involved in handling these products had no personal protection equipment (PPE), 62% of them reported ‘feeling ill” while they handled pesticides. The most common symptoms were: headache, sickness, vomiting, vertigo, skin rash, and blurred vision.The literature indicates that subclinical symptoms should be considered as early signs of poisoningVestibular system alterations detected by tests are not reported in the literature. Therefore, only after vestibular examination according to the parameters defined for this study, will questions be answered or further research recommended.This is a cross-sectional historical cohort study developed based on the vestibular function analysis of 18 rural workers from the municipality of Teresópolis, in the state of Rio de Janeiro, Brazil, located more specifically in the Córrego das Pedras and Baú regions. Ages varied between 16 and 59 years. Gender was not specifically targeted as a variable. Exposure time was calculated based on the number of years the workers have been active for.Inclusion criteria were complaints of dizziness and occupational exposure to organophosphorous compounds; exclusion criteria were smoking, alcohol and drug abuse, metabolic, hormonal and neck disorders, as these factors are the main points where vestibular disorders are originated.No specific age range was studied. The groups were not divided into age groups, as no data was found in the literature on worker age and normality patterns. Gender was not deemed as a relevant variable, as we followed the guidelines published by Mangabeira Albernaz, Ganança et al. (1986)Three questionnaires were given to participants with the purpose of gathering information. They contained questions pertaining to the inclusion/exclusion criteria as specified previously.Questionnaire I contained questions on ear anatomic and physiological disorders; auditory and vestibular symptoms; health in general, specifically addressing exclusion criteria and general information on the type of work they did. Questionnaires II and III contained questions on how work was done and its relations with dizziness.All workers went through otorhinolaryngological, audiological, and vestibular examination.In order to characterize normal hearing thresholds in adults, we adopted the hearing normality criteria for various ages defined by Glorig, Davis (1961)Vestibular examination was done using program Contronics under the principles defined by Mangabeira Albernaz, Ganança et al. (1986)The study considered ethical aspects as recommended by Resolution 196/96 on research with human beings and included, among others, the obtention of free informed consent from participants. We ensured that individual participation would not lead to physical, psychical, moral, intellectual, social, cultural, or spiritual damage. This study was approved by our institution under permit 38/2005.Eighteen rural workers were interviewed. Most of them were women, as seen in The most broadly used pesticides were Manzate (54.2%) - toxicological rating III, followed by Folidol (33.3%), Tamaron (27.1%), and Gramoxone (25%), all under toxicological rating I, considered highly toxic (Decree 98.816/90 from Law 7802/89).Audiometric tests found that 11 (61.14%) subjects had normal hearing, 7 (38.8%) had altered tone audiometry test results - 4 (22.22%) subjects had decreased performance at 6000Hz and 8000Hz and 3 (16.67%) had sensorineural hearing loss. All subjects had speech audiometry within normal ranges.Seven subjects (38.9%) with normal tone audiometry tests were aged between 16-35 years and five subjects with altered tone audiometry test results  were aged between 46-59 years.Only two subjects (11.11%) had bilateral hearing loss.Eighteen subjects underwent VENG, and 16 (88.8%) were found to have peripheral irritative vestibular syndrome, 11 of whom due to altered nystagmus test results and/or positional vertigo and 9 due to altered results in the caloric test, presenting hyperreflexia in absolute values as seen in In terms of the aspects connected to the daily lives of the workers, we can see on Thirteen workers (72.25%) answered that the chemical products they use at work affect their balance, and 16 (88.9%) believe their work poses health risks. This last question did not directly relate hazardous working conditions to the use of pesticides.In contrast, the number of years on the job on the job and the daily time of exposure to pesticides interfered with test results, as 8 subjects (44.5%) with altered test results had been working for over 21 years and 13 subjects (72.3%) with altered VENG worked for more than 10 hours a day.This scenario is worsened when we see that the use of personal protection equipment (PPE) could make a difference, as 77.8% of the subjects failed to use PPE and had altered vector electronystagmography results.Faria et al. (2004)Audiometric findings indicate that noise and chemical poisoning induced hearing loss are very similar to presbycusis. Hearing loss differential diagnosis can be done only through careful interview and clinical examination. Diagnosis can be quite challenging, as causes for disease often overlap.Most subjects with normal and altered test results had been doing this job for 21-45 years. This indicates that hearing health does not vary according to time of exposure to pesticides. The same trend was observed when we looked at audiometric tests and number of hours individuals worked per day.Failure to wear PPE  such as masks, gloves, and proper apparel to handle pesticides may be harmful to hearing. Teixeira, Augusto et al. (2003)Castro et al. (2005)All 18 rural workers that participated in our study had dizziness spells, 7 (38.9%) had hearing loss and 16 (88.8%) had vestibular disorders. Whenever reliable data on the possible impact a harmful substance may have on the environment or upon human health is available, the principle of precautionDizziness stood out in our study as one of the clinical manifestations connected to pesticide exposure that must be assessed and detected during clinical interviews with rural workers and properly considered by the health care teams in charge of serving rural communities.Workers are aware of the dangers pesticides offer to their health and the environment. They must therefore be trained on how to adequately use pesticides and informed of other means of dealing with their crops without the aid of pesticides.The rural population exposed to pesticides is not limited to farmers. It also includes their families who live inside plantation areas and local communities. More research should thus be conducted on pesticide exposure assessment and the risk associated with these products. The literature sees dizziness as a subclinical symptom of acute and long term pesticide poisoning, a condition that adversely affects workers and renders them unable to work and live well with their families, deteriorating both health and quality of life.Considering the mean time for which the workers assessed in this study were exposed to pesticides and excluding the various aspects that contribute or trigger dizziness spells such as age, metabolic and hormonal disorders, social habits, among others, the data gathered in this study indicate that organophosphorous pesticides promote disorders in the vestibular and hearing systems, apart from potentially increasing the risk of neuroototoxic events.The effects organophosphorous pesticides have upon human health encompass a variety of signs and symptoms that go way beyond clinical visibility, and thus call for a complex approach to health care so as to ensure treatment and prevention in terms of environmental and personal care in all its spheres.


Hearing loss is more prevalent than other disorders found at birth. Efforts have been put up towards the early identification and treatment of hearing loss by means of neonatal hearing screening programs.prospective study with the goal of characterizing the process of implementing a Neonatal Auditory Screening Program (NASP) at a University Hospital. To analyze hearing loss diagnostic investigations in newborns, and to present proposals for NASP improvement.we studied newborns (NB) submitted to Newborn Auditory Screening (NAS) by transient evoked otoacoustic emissions (TEOE), cochlear-eyelid reflex (CER) and Brainstem Evoked Auditory Potential (BEAP).we tested 625 children. In the first stage, 458 NBs passed and 155 failed. 122 NBs returned to the second stage, and 8 underwent it because they were positive for HL risk factors. 12 NBs (1.9%) were referred for diagnostic investigation. Of the 5 who returned for the BAEP, we observed HL in two NBs.the program tested 81.7% of the candidates. The program compliance rate was of 68.2%. In the first stage, 26.7% of the NBs failed. The program is being implemented and requires constant analyzes of its difficulties, aiming at solving them in order to turn the Universal Newborn Auditory Screening into reality. Today, because of the magnitude of the losses caused by hearing impairment, this topic has received broad attention from health care authorities throughout the world. For quite some time now, the need for early detection and proper treatment is advocated, since the diagnosis of this disorder, usually late, around the third year of life,The early treatment of hearing loss brings known benefits, through the use of hearing aids, all the way to a cochlear implant, in order to enhance normal language development, especially when done before six months of life.In Brazil, hearing loss early detection programs have been carried out in maternities of 22 states, following international recommendations, in a total of 237 services registered at the Grupo de Apoio à Triagem Auditiva Neonatal Universal  and some cities which have legislation establishing that it is mandatory to perform hearing screening in all newborns.4The present investigation aims at characterizing the Neonatal Auditory Screening Program in a University Hospital, analyze the diagnostic investigation of hearing loss in newborns and present proposals to enhance neonatal hearing screening.MATERIALS AND METHODSThis study was a cross-sectional contemporary cohort. The subjects were all newborns (NB) submitted to hearing screening at the hospital from March 01 to August, 31 of 2005. Three NBs were taken off the study because it was not possible to see their charts.The study project was approved by the Ethics Committee, on December 13, 2004, under protocol # 324/04. Parents or guardians had to sign a free and informed consent form.In order to assess hearing acuity, we used the transient otoacoustic emissions hearing accuracy device, from Madsen, Accu Screen Pro T model, produced by GN Otometrics, near the hospital discharge hour of the NBs admitted to joint quarters or after clinical stabilization. The behavioral assessment was carried out by means of studying the cochleopalpebral reflex (CPR), using an agogo as an instrument, at the time of the emissions test.In this first step, the in-hospital tests were carried out from Monday through Friday. The untested NBs and those discharged on the weekends and/or holidays were referred to outpatient evaluation between two and four weeks of life. The tests were conducted by speech and hearing therapists. The probe for otoacoustic emissions capture was coupled to the newborns' outer ears, preferably during their natural sleep and after breastfeeding. Should the test yield a hearing alteration, it was repeated on the second stage of the program, between 7 and 15 days after hospital discharge.On the second stage, the middle ear was assessed by means of the tympanometric curve in those patients with altered screening tests. These patients would be referred to medical assessment for new tests.When indicated, the NB would go through a third investigation stage, being then assessed by Brainstem Evoked Auditory Potential (BEAP), by an interacoustics ABR EP15 system.Statistical AnalysisThe data collected was organized and analyzed by the Epidata 3.0® and Epi-Info 6.0® software. For the numerical variables, we calculated the absolute values and the variability and position descriptive measures (mean and mean standard deviation). In order to check the significance, we used the X2 (α < 5%) test.During the study period, we had 774 live births in the maternity. We included in the study two newborns from home deliveries who were admitted after birth. One NB was transferred to another hospital. Four NBs died in the obstetric center and six died in the neonatal unit. Thus, a total of 765 NBs were eligible for neonatal auditory screening. On Of all the 625 patients tested, 595 (95.2%) underwent auditory testing by TOAE during their hospital stay. 30 NBs who returned to the outpatient ward were tested (4.8%). Of the 765 NBs eligible to the auditory test, 140 (18.3%) were not tested during the hospital stay and did not return to the outpatient test.In the first Newborn Auditory Screening - NAS, the TOAE tests were normal - negative/passed - in 458 NBs (73.3%) and altered - positive/failed - in 167 (26.7%). The Cochleopalpebral reflex - CPR was altered in 38 NBs (6.1%) .Table 1DThe unilateral failure type was identified in 97 NBs (62.2%), while bilateral failure happened in 58 (37.4%) from the total of 155 emission tests that failed.For the second NAS stage, 122 NBs participated and 8 of them who passed the first stage tests returned for having risk factors. In the retest, 114 NBs passed the TOAEs and 8 failed, and of these 5 (62.5%) failed bilaterally. The CPR was absent in 8 NBs .Table 2DTympanometry was normal in 12 patients who failed the retest. These 12 patients were referred to the otorhinolaryngology department and for BAEP evaluation. The NBs born in the first 6 months of implantation, when we still did not have the test and who were waiting for it, were called in. Six NBs were located by their phone numbers recorded in their charts. One of the NBs is being followed up in another municipality. Five children came for investigation and BAEP. In these we confirmed the hearing loss in two, representing 0.32% from the total population studied .Chart 1FThe main diagnosis found among the 57 NBs admitted to the ICU are represented on The risk indicators for congenital hearing loss seen in the study were analyzed separately, according to The association of risk factors together showed a trend towards statistical significance to present a TOAE failure .There was no association between the use of ototoxic medication and the rate of TOAE failures (p>0.05) , Table 6,According to the references and the quality indicators defined by the JCIH in 2000, in order for a NAS program to be universal, within six months of implementation it must reach a minimum of 95% of infants assessed during post-partum admission, or before one month of life.The screening was carried out in 595 NBs (95.2%) during the stay in joint quarters at the Neonatal Unit. 17.96% of the NBs returned for outpatient testing, 30 of the 167 NBs expected. The rate of “no shows” at the outpatient ward screening first stage was of 82%. A study carried out in Malaysia, by Mukari et al.For the narrow scope in the first maternity test, Mukari et al. relate to hospital discharge before 24 hours after birth to the possibility of return after discharge, and the test performance was no longer a priority during hospital stay; and the fact that the examiners worked from Monday to Friday, and the test was not performed in those children to be discharged during the weekend.On the NAS first stage, in this service, 458 NBs (73.3%) passed the TOAE and CPR tests and 167 (26.7%) failed, as per described on As we analyzed the failures reported by Wroblewska-Seniuk et al. in 2005, we observed a positive test in 16.28% of the total of 5,601 NBs studied. Of these, 61.5% failed unilaterally on the TOAE test.It is a fact that the high rate of TOAE false-positives represent a disadvantage of this type of test. This NAS had 16.9% of false-positive emissions on the first stage of the assessment. Marone et al., 2005, reported 15.5% of false positive tests.,,The short hospital stay period after birth can raise the false-positive rate. The presence of amniotic liquid and caseous vernix in the auditory canal may impair sound transmission and cause failure in the emissions test.,All the children with the first TOAE positive test and or without CPR, were referred for retesting in the lab. And, following the guidelines suggested by the JCIH, the NBs with some high risk indication concerning hearing loss, because of the risk of progressive loss or a loss of late onset, were referred for follow up every six months for three years.,For the second stage with 122 children, 114 were among the 167 NBs who failed the first stage, representing 68.2% of program compliance, that is, the children who failed the first stage and returned for retesting. This value is similar to the 73.1% described in the Durante et al study and more expressive than the 41.8% compliance observed by Korres et al. at the end of the fifth year of universal screening with TOAE only (Chart 2).,,On the second screening stage, 6.6% of the children failed the TOAE test. In the studies reported above, the authors observed higher failure rates in the retest with TOAE, from 23.81 to 39.91%.The behavioral assessment is considered an important part of the investigation as it provides information of how auditory resources are used by the child and for assessing central auditory pathways. The presence of behavioral information suggests integrity of the hearing pathways and no severe hearing loss.In the present investigation, CPR was absent in 6.1% of the time in the first screening stage and in 6.5% on the second. In the study carried out by Pádua et al. they found an index of 1.8% of CPR alterations at the UNAS.12 children were referred for diagnostic investigation follow up, representing 1.9% of the NBs tested in the Neonatal Auditory Screening Program in six months and 1.6% of the live births eligible for hearing screening (765). The NAS quality indicator proposed by the JCIH indicates that at the end of the first year of the program, less than 4% of the NBs screened should be referred as a goal to avoid high false-positive rates.On the 12 children referred for the third stage of the follow up, we see the presence of at least one risk factor in 11 of them .187 newborns with risk factors for hearing loss were identified, representing 29.92% of the sample. Of these, 37 newborns failed TOAEs . The ratNóbrega et al. described congenital rubella as one of the main etiologies in children, even showing a high percentage of loss for unknown cause.Maternal infection by HIV can also be considered a risk factor because of vertical transmission.19One important factor observed in the sample is the presence of hearing loss in relatives of the first to the third grades (6.1%). Hereditary can represent up to 50% of hearing loss in childhood.It is estimated that the prevalence of hearing deficit in children with past history of ICU stay increase in 6 fold, because of the higher risks of comorbidities and complications.Of the 12 children referred for a third stage investigation, five underwent brainstem audiometry test. Results were altered in two children, representing 0.32% of the study population (Chart 2). Hall et al., 2004, studying the combination of TOAE with BAEP, describing a 2% rate of positive screening.23The goal established by the JCIH is reaching diagnosis before 3 months and perform intervention by 6 months of age. In the study program, hearing loss diagnosis of the two children was carried out between 7 and 10 months of age. They are being evaluated by the multidisciplinary auditory development follow up program for proper treatment. The children without BAEP test are being looked for, for testing.After the screening results in 81.7% of the NBs eligible, we tried to identify the factors that could justify them. Checking all the births at the maternity from the Obstetric Center Records and crossing such data with those from NBs who had been submitted to screening, we noticed that of the births which happened on Thursdays and Fridays of NBs who did not undergo TOAEs before hospital discharge, 57.6% were born in these two days of the week. Of these, 20.7% came from other towns.,Korres et al. (2006) suggest the factors that can make an auditory screening program a success. During the first three years of the program implementation in Athens, the authors identified the probable causes of narrow scope. In the two following years, they continued to enhance the program, and currently, the program reached the metrics proposed by the JCIH. The first testing before discharge went from 58.9 to 96.3% and the return for tests in the outpatient ward increased from 27.8 to 41.8%. The authors relate the good results especially to the fact that NBs are being discharged between four and five days of life. The first test is carried out before 24 hours, and when it fails, it is repeated many times until the day of discharge. The program also includes tests during the 7 days of the week, training of all the professionals involved with the care of pregnant women, from prenatal care to the delivery, to the care given to the newborn all the way to their discharge and follow up. NBs who failed had their testing repeated many times until the time they went home is stressed by the authors as the main factor responsible for the program to have reached only 2.1% of fails in the first OAE. These NBs who could effectively have hearing loss and the need to follow the program were more easily followed in the continuation of the investigation and intervention. Another thing that was stressed was to alert the parents regarding the probable risk of hearing deficit in their children, since the test was carried out many times, engaging them even more emphatically regarding follow up after going home.,Korres et al.During the study, the NAS in this university hospital completed one year in the beginning of the implementation. Some improvements started after focusing more intensively on the attempt to guarantee the performance of these tests before maternity discharge. Other improvements are still to be implemented in order to increase the rates of compliance and reduce the rates of false-positive.The joint quarters team and the Neonatal Unit is more prepared to identify the NBs who have not yet been tested. The members of the medical team are alert as they discharge these children. As far as weekends and holidays are concerned, there is the need to create mechanisms for NBs with discharge scheduled for these days, either be tested before, or that the tests be performed even during these days by appointed professionals.At the time of hospital discharge, we suggest to identify the charts of NBs who suffered the test and it was normal and the ones who failed, and who should return. The ones who were not registered, therefore were not submitted to the first TOAE, and are being discharged, may be identified later on, for active search in case they miss their return scheduled to the outpatient ward.In order to better educate parents at the time of discharge, we suggest the creation of an educational card. In such a card, there must be the identification, data and time of hospital discharge; date, time and place of return; and also the telephone number of the program for the possibility of a new scheduling of the return visit when necessary.The disclosure to the community by means of communication, the alert about the need to undergo hearing deficit screening as early as possible and the involvement of all health care professionals from a primary level, during prenatal care and during pediatric care, are strategies to educate parents regarding the importance of hearing screening. Parents may become inspectors of the program's effectiveness, as it happens today with the foot test.Because of its repercussion on citizenship, hearing loss represents a public health problem. Health care authorities have the responsibility of stimulating the implementation of programs in the many cities, creating points of reference for easier access by the population. And after implementing them, means must be created to assure their real effectiveness.The data from this study are related to the first six months of the program. There is the need to update the data in the program's sequence and check for the effective improvement on test rates in its many stages. The implementation of a neonatal hearing screening in a university hospital, which serves only the public health care system, bears innumerous difficulties. The involvement of all the members of the multidisciplinary team is paramount.The first 6 months of implementation of the Neonatal Hearing Screening Program in the University Hospital have an engaging rate of 81.7% of the eligible newborns. The compliance rate, in other words, return of newborns with TOAE failure was of 68.2%. Of the NBs tested, 1.9% required investigation by the BAEP and 0.32% had hearing loss.Some important things were noticed at the time of implementation: team dedication and commitment, disclosure and family education, and their interest in such a program.In order to improve the NAS program scope we propose: that the tests be repeated before hospital discharge, tests be carried out during the weekends and holidays, a greater integration of the health care team, distribute explanatory cards to the parents during hospital stay, better information on the press and greater governmental action in order to divulge NAS.The program is still in its implementation phase; the difficulties found and others that will come must be constantly analyzed and the solutions put into practice in order to make the screening program truly universal.