Early screening and detection of individuals at high risk of high-frequency hearing loss and identification of risk factors are critical to reduce the prevalence at community level. However, unlike those for individuals facing occupational auditory hazards, a limited number of hearing loss screening models have been developed for community residents. Therefore, this study used lasso regression with 10-fold cross-validation for feature selection and model construction on 38 questionnaire-based variables of 4010 subjects and applied the model to training and testing cohorts to obtain a risk score. The model achieved an area under the curve (AUC) of 0.844 in the model validation stage and individuals’ risk scores were subsequently stratified into low-, medium-, and high-risk categories. A total of 92.79% (1094/1179) of subjects in the high-risk category were confirmed to have hearing loss by audiometry test, which was 3.7 times higher than that in the low-risk group . Half of the key indicators were related to modifiable contexts, and they were identified as significantly associated with the incident hearing loss. These results demonstrated that the developed model would be feasible to identify residents at high risk of hearing loss via regular community-level health examinations and detecting individualized risk factors, and eventually provide precision interventions. Hearing loss is the fourth leading cause of disability worldwide . AccordiEtiologies of hearing loss are multifactorial, and the common risk factors include genetic variation, noise exposure, ototoxic drugs, aging, and lifestyle factors . There iHearing loss usually starts from high-frequency hearing issues and then degenerates to language frequency disability; high-frequency hearing loss screening is the key to early detection of hearing issues . The WHOMany researchers from relevant study fields have developed screening tools for hearing-impaired listeners, including scales and technology-based hearing screening tools ,7. Most For the purpose of screening or prediction, most previous hearing loss prediction models typically focused on professional workers who have occupational noise exposures, or individuals afflicted with a sudden sensorineural hearing loss (SSHL). There are several prediction models for evaluating such hearing loss risk. One model was developed for the prediction of hearing loss in individuals facing occupational auditory hazards, and it showed a discriminatory accuracy of 78.2% . AnotherTherefore, the aim of this study was to establish a high-frequency hearing loss risk prediction model for adults residing in the community, by using data from 4010 residents of Zhejiang province; to apply the built model to the training and testing cohorts and obtain risk scores that describe an individual’s probability of receiving a diagnosis of high-frequency hearing loss; and to stratify residents into low-, medium-, and high-risk categories based on these risk scores. We further aimed to identify modifiable risk factors for hearing loss, and to provide guidance for screening of hearing loss and intervention of high-risk categories.The multistage, stratified cluster random sampling method was used to examine the health of residents in Zhejiang province at five hospitals and two community health centers from September 2016 to June 2018. After providing informed consent, 4010 residents participated in the survey and completed the questionnaire survey and audiometry test. The inclusion criteria are shown in All pure-tone air-conduction hearing thresholds were measured by professional medical staff using audiometers  and TDH-39 headphones  in a well-ventilated listening chamber with a background noise <30 dB(A). Subjects were recommended to stay away from noisy environments for more than 12 h before the audiometry test in order to improve accuracy. In the intensity range of −10 to 110 dB, pure-tone air conduction hearing thresholds were tested in both ears of the participants at frequencies of 0.125, 0.25, 0.5, 1, 2, 3, 4, 6, and 8 kHz . ParticiThe questionnaire survey and audiometry test were conducted on the same day. The original questionnaire was first evaluated and revised through expert consultation. After the formal questionnaire was generated, a pilot survey involving 926 participants was first conducted to verify the questionnaire. Before the implement of the pilot survey, all investigators had been well trained to ensure the quality of data collection. As a result, they were able to provide accurate explanations of both items and options in a face-to-face interview, thereby minimizing the respondent’s bias. During the pilot survey, the developed questionnaire reached a Cronbach α coefficient of 0.753, and a KMO value of 0.794.The questionnaire included six parts: (1) demographics and social determinants ; (2) symptom histories ; (3) disease conditions ; (4) behavioral factors ; (5) environmental exposure ; and (6) hearing cognitive parameters . The details of the questionnaire items and the answers given by the respondents are shown in Previous research has identified a broad range of risk factors for hearing loss which can be classified into three groups , includiIt should be noted that symptom histories  were measured by the frequency of their self-reported occurrence within the past year, while disease histories  were determined based on participants’ self-reported current or historical physician diagnosis. In terms of behavioral conditions, most variables were measured as current or past-1-year conditions, while smoking/drinking status were measured as none or little smoking/drinking, former smoking/drinking, or current smoking/drinking . In addip-value of <0.05 was considered for statistical significance. After applying this criterion, 36 out of 38 features were selected  to enter the survey data and to check and correct errors. Before feature selection, in order to ensure the authenticity of the data, the samples corresponding to the missing data were directly deleted. The cases were identified according to audiometry test results. Chi-squared test was adopted to evaluate the difference between various covariates between cases and controls, where all reported probability values were two-tailed, and a selected .n = 4010) was randomly divided into a training cohort (n = 2667) and a testing cohort (n = 1343) at a ratio of 2/3:1/3; 36 features were selected, and the least absolute shrinkage and selection operator (lasso) regression with 10-fold cross-validation and penalty was used to construct a hearing loss prediction model by “glmnet” R software package. Lasso regression is a widely used machine learning algorithm; compared with traditional logistic regression, it uses a penalty term, which can actively select impactful parameters from a large set of potentially multicollinear variables in the regression, helping to reduce prediction errors [The cohort (n errors .n = 4010) from low risk to high risk according to the risk score, and divided them into three groups: low-, medium-, and high-risk group. Performance of the model was investigated within each risk category in terms of positive predictive value (PPV). All statistical analyses were performed using R V.4.0.  of whom were diagnosed with high-frequency hearing loss. Among these 2232 cases, 55.5% (1238/2232) were male and 26.3% (586/2232) received junior high school education. The major diseases included current hypertension  and diabetes mellitus . In terms of lifestyles, 25.0% (558/2232) of patients with hearing loss smoked, 21.1% (470/2232) consumed alcohol, 45.7% (1019/2232) barely exercised, and 63.2% (1410/2232) rarely worked in a noise exposure environment. The remaining descriptive statistics for all other predictors are available in Application of the lasso regression to the data showed that our prediction model achieved a fitted AUC of 0.864  0.850–0.878) in the training cohort and a predicted AUC of 0.844 (95% CI 0.823–0.865) in the testing cohort . Based oA total of 22 impactful features were identified as the final predictors of the model, including 20 predictors that were related to modifiable contexts and two nonmodifiable predictors. The estimated coefficients are listed in To further explore the distribution of captured lifestyles across the three risk categories, we grouped individuals across the spectrum of risk scores and calculated the prevalence of certain lifestyles under each risk bin. As shown in Age was recognized as an impactful demographic feature in our high-frequency hearing loss prediction model. Among seven distinct age groups included in this study, young individuals mainly constituted the low-risk group, whereas older individuals were concentrated in the high-risk group . RegardiThe individuals diagnosed with four common diseases  showed a dramatic increase in hearing loss risk score from the range corresponding to the low-risk group to that corresponding to the high-risk group . More thThe high-frequency hearing loss screening model developed in this study achieved an AUC of 0.844 in the model validation stage, indicating that it has a good discriminatory ability and could be potentially applied to community residents living in southeast China, using their self-reported predictors. Using this prediction model, we classified the population into high-, medium-, and low-risk categories. The PPV of the high-risk group was 92.79%, which was 3.7 times higher than that of the low-risk group (25.18%), and half of the predictors were related to modifiable context. It is believed that the developed model could facilitate initial screening and help identify individuals at a high risk of hearing loss for the implementation of precision intervention.Social determinants are associated with health from various perspectives . UnderstSmoking, alcohol consumption, and exercise habits, which are shaped by social and economic factors, are impactful behavioral drivers of hearing loss . SmokingIn addition, high stress in daily life was recognized as a predictor of hearing loss in our screening model, which may also have interacted with material and interpersonal social determinants, triggering unhealthy lifestyle choices such as excessive alcohol consumption and visceral obesity , and eveAge and gender were nonmodifiable predictors in our prediction model. Hearing decline along with aging is generally attributed to progressive peripheral degeneration, including the degeneration of cochlear sensory hair cells, stria vascularis, and spiral ganglion neurons leading to the disability of sound afferent . Our preIn our study, hypertension was a predictor, and it may increase the risk of hearing loss by reducing blood supply to the stria vascularis, which is in the lateral cochlear wall and is responsible for sending auditory signals from the cochlea to the central nervous system. Vascular supply to the stria vascularis is derived from terminal arteries with no collateral supply. Therefore, the stria vascularis is particularly sensitive to events that compromise vascular supply, with animal studies showing reduced end cochlear potential and hearing loss occurring promptly after an anoxic event . DiabeteSymptom histories, such as tinnitus history and aural fullness history, were also recognized as modifiable indicators of hearing loss in our model. Tinnitus, the perceived sensation of a sound that has no external source , could bTo date, prediction models for high-frequency hearing loss in the general population are still lacking. Unlike our prediction model, most existing hearing loss screening tools  only focus on evaluating the severe outcomes and functional status of hearing deficit and fail to identify individualized risk factors of hearing loss. It was reported that under different circumstances, the sensitivity of HHIE (the instrument for elderly) could range from 24% to 100% ,44. On tAt present, hearing loss prediction models developed for community residents is limited; it is believed that our screening model can help improve the delivery of health care services at multiple levels. First, compared with existing tools, it has the advantages of low cost, ease of implementation, and no need for supporting equipment. Second, the model can stratify the population based on the obtained risk score of hearing loss, which can help to target high-risk populations and facilitate decision-making and implementation of interventions. Third, the model, as a screening tool, can help primary care physicians identify individuals who should be referred to hospitals for audiometry tests to facilitate the diagnosis of hearing loss (high risk vs. low risk). Most importantly, the model can identify essential and critical modifiable risk factors for each community resident. On this basis, healthcare workers could design personalized health education and promotion programs to improve individuals’ self-consciousness capabilities in relation to high-frequency hearing loss.Our study also has several limitations. First, self-reported noise exposure information was obtained, which could cause potential bias and misclassification. Second, the model’s performance should be further validated in an independent cohort, ideally consisting of a population from different areas of China to guarantee the model’s generalizability to a larger number of people. Third, the risk factors involved in this study may not be comprehensive enough, and the built model’s screening ability may be further improved after the collection of more valuable predictors.This study developed and validated a risk prediction model to identify high-risk groups of community residents with high-frequency hearing loss. The model consisted of simple questionnaire survey items and showed the risk score of individual factors. The risk screening model can easily identify people at high risk of hearing loss and the unique risk factors of each community resident. On this basis, medical workers could design personalized health education and promotion programs to improve individuals’ self-consciousness and self-care capabilities in relation to high-frequency hearing loss.


Is hearing loss associated with lower levels of physical activity?In this cross-sectional analysis of 2490 US adults aged 30 to 69 years, worse hearing, as defined by pure-tone audiometry, was associated with lower objectively assessed physical activity levels starting at a 15-dB hearing level threshold.These findings suggest that lower levels of physical activity may be one of the mechanisms explaining the adverse associations between hearing loss and several health outcomes, such as cognition, physical functioning, and hospitalizations. This cross-sectional study evaluates whether hearing loss is associated with lower levels of physical activity. Lower physical activity (PA) levels have been proposed as a mechanism to explain the association between hearing loss and various adverse outcomes of aging. But whether hearing loss is associated with lower PA is poorly understood.To evaluate whether hearing loss is associated with lower levels of PA.This cross-sectional analysis was conducted in a representative sample of US adults (aged 30-69 years) who participated in the 2011-2012 cycle of the National Health and Nutrition Examination Survey (NHANES). Data analysis was conducted from July to September 2021.Hearing was assessed, in dB, by pure-tone audiometry and summarized as the better hearing ear’s pure-tone average (BPTA) of 4 speech frequencies ; a higher BTPA indicates worse hearing. Hearing was also categorized into normal (BPTA ≤25 dB), mild hearing impairment (>25 to 40 dB), and moderate or greater hearing impairment (>40 dB).Total 24-hour movement activity was measured using wrist accelerometers worn at all times for 7 consecutive days and summarized in monitor-independent movement summary units (MIMS) units (higher MIMS units indicate more movement). The association between BPTA and MIMS units was evaluated using a multivariable-adjusted (demographic characteristics and chronic conditions) piecewise spline regression (knot at 15 dB). Mean differences in MIMS units across hearing categories compared with normal hearing were estimated.The study included 2490 participants . Before the knot at 15 dB, we found that each 10-dB higher BPTA was associated with 860.4  higher MIMS units. Conversely, after the knot, each 10-dB higher BPTA was associated with 458.6  lower MIMS units. The difference in MIMS units across hearing categories was not statistically significant.In this cross-sectional study, in the range of hearing sensitivity of BPTA of 15 dB or greater, worse hearing was associated with lower MIMS units. Lower PA may be a mechanism contributing to the association between hearing impairment and adverse health. Higher levels of physical activity are associated with a lower cardiovascular disease risk, increased longevity, and higher quality of life.9 Previous studies suggest that hearing loss may lead to adverse health outcomes, including faster decline in cognitive function, slower gait speed, and poorer balance at older ages.13 One mechanism that may explain these associations is lower levels of physical activity among those with impaired hearing.Hearing loss is common and frequently undetected among middle-age and older adults, affecting approximately one-fifth of middle-aged adults and nearly two-thirds of adults older than 65 years.16 While these previous studies have used accelerometry measures to derive metrics of physical activity, only 1 study17 has used physical activity metrics that go beyond time spent at different intensities . Moreover, accelerometry-derived metrics typically are device specific, making it difficult to contrast findings from studies that use different accelerometer devices. Hence, novel physical activity metrics have been developed leveraging accelerometry-collected data that are not specific, such as Monitor-Independent Movement Summary (MIMS) units.18 This study aimed to investigate the association between hearing loss measured by pure-tone audiometry and physical activity summarized using MIMS units among the nationally representative National Health and Nutrition Examination Survey (NHANES) sample of adult participants of the 2011-2012 cycle. We hypothesized that participants with higher hearing thresholds in the audiometry test  would have lower levels of physical activity.Previous research has shown that adults with hearing loss perform less physical activity, objectively measured, than those without hearing loss.19 NHANES participants completed an in-person home interview to provide demographic, socioeconomic, and health-related information. Physical examinations were conducted in a mobile examination center. During the examination, participants were invited to wear a physical activity monitor and complete a pure-tone audiometric assessment. We excluded 63 participants who reported hearing aid use because hearing aid use may modify the association between hearing loss and physical activity. We also excluded 444 participants who did not have physical activity data, and 15 participants who had missing information on the covariates of interest. The final analytic sample included 2490 adults aged 30 to 69 years who completed the hearing and physical activity assessments.Our study population included 3012 participants, aged 30 to 69 years, recruited to the 2011-2012 NHANES cycle who completed the audiometric assessment; this NHANES cycle was selected because, of the recent cycles when wrist accelerometry data were collected, pure-tone audiometry was obtained only in 2011-2012. NHANES uses a complex, multistage probability design to oversample racial and ethnic minority groups and individuals with lower income and obtain a nationally representative group of non-institutionalized US residents.STROBE) reporting guidelines for cross-sectional studies.20The protocol for NHANES was approved by the National Center for Health Statistics institutional review board, and all participants provided written informed consent. Per the Common Rule, institutional review board approval for this analysis was exempt, as NHANES data are deidentified and publicly available. This study followed the Strengthening the Reporting of Observational Studies in Epidemiology . NHANES participants were asked to wear the device on the nondominant wrist for 7 consecutive complete days (examination day [day 1] and requested return date [day 9] were not considered), including during water-based activities . The ActiGraph contains a triaxial accelerometer that was programmed to collect data at 80 Hz.22 and in adherence with the NHANES accelerometry protocol,23 days in which wear time was incomplete (<1440 minutes) or with 17 or more hours of sleep  were classified as nonvalid and excluded. All participants with at least 1 day of valid wear were included in our analyses since previous studies have determined that 1 day is sufficient for population-level analyses.24To reduce measurement error, as is commonly done in research using data from wrist-worn accelerometers,25 in dB hearing level (HL) at different frequencies for each ear separately. We calculated a 4-frequency  pure-tone average for each ear and used the better-hearing ear’s pure-tone average (BPTA) as a continuous measure for analysis. Consistent with previous literature, we further categorized participants hearing status into normal hearing (BPTA ≤25 dB HL), mild hearing loss (BPTA >25 to 40 dB HL), and moderate or greater hearing loss (BPTA >40 dB HL).26Hearing was assessed by trained NHANES staff using pure-tone audiometry. The tests were performed in a sound-attenuated booth with a calibrated audiometer in the mobile examination center. Air conduction hearing thresholds were identified using best-practice threshold seeking methods1c [HbA1c] ≥6.5% ), hypertension , smoking status , heart attack (ever been told by a doctor you had a heart attack), stroke (ever been told by a doctor you had a stroke), and heart failure (ever been told by a doctor you had heart failure). We selected this set of covariates for adjustment in our analysis based on their documented associations with both hearing and physical activity, making them potential confounders of our association of interest.Our demographic covariates of interest included age, sex, race and ethnicity , education , marital status (married vs not) and employment status (currently working vs not). We also included some anthropometric measures and chronic conditions, such as body mass index , diabetes . We built 2 models: model 1, adjusted for age, sex, race and ethnicity, education, marital status, employment status, BMI, diabetes, hypertension, and smoking status. Model 2 additionally adjusted for stroke, heart attack, and heart failure.In descriptive analyses, to compare demographic and medical characteristics across the hearing loss groups, we used analysis of variance and χInitial exploratory data analysis, via 2-way plots with locally weighted smoothing curves, revealed a nonlinear association between MIMS units and BPTA; thus, we introduced a 2-piece linear spline term for BPTA with the knot at 15 dB HL, the value at which we observed a change in the curve. We assessed the mean differences in MIMS units per 10-dB HL higher BPTA (worse hearing) using 2-piece linear spline regression models. We also estimated the mean difference in MIMS units across hearing loss groups .27 We also examined a knot at 25 dB HL to match former WHO guidelines and the previous literature.9 Moreover, we created a model with restricted cubic spline terms that model this association more flexibly. Additionally, we created 4 activity groups using age- and sex-specific quartiles of MIMS units previously developed using NHANES data,21 and used ordinal logistic regression models to evaluate whether participants with hearing loss had a higher odds of being in a lower activity group. All analyses were conducted using Stata version 15 (StataCorp), and the svy commands were used to account for the complex sampling design of NHANES. We considered P < .05 statistically significant, and all tests were 2-tailed.In sensitivity analyses, we examined changing the knot in our spline regression to 20 dB HL (rather than 15 dB HL) to match the current World Health Organization (WHO) thresholds for distinguishing normal hearing from mild hearing loss.Among the 2490 participants, 191 (7.7%) had mild, moderate, or severe hearing loss. The age range was 30 to 69 years , 1248  were female; 900 , White; 695 , Non-Hispanic Black; and 267 , Mexican American. In weighted, unadjusted comparisons , particiIn our 2-piece linear spline adjusted regression analysis , we obseThe mean differences in MIMS units across hearing loss groups are provided in In changing the knot to 20 or 25 dB HL . After 15 dB HL knot, however, each 10-dB HL higher BPTA had higher odds of being in a lower physical activity group, but this finding was not statistically significant . Similarly, mild and moderate or greater hearing loss had non–statistically significantly higher odds of being in a lower activity group .In a weighted analysis representative of the US population aged 30 to 69 years, higher BPTA was associated with lower MIMS units. Our findings from secondary analyses that compared differences in mean MIMS units and odds of being in lower activity categories across hearing loss categories supported our hypothesis that participants with poorer hearing had lower levels of physical activity. While these results were not statistically significant, it is likely that this is due to a relatively low number of participants in the mild and moderate or greater hearing loss groups.17 who recently evaluated this association using data from participants aged 60 to 69 years from the 2003-2004 NHANES cycle. While both analyses arrived at similar conclusions, our sample included a wider age range and used novel activity metrics that are monitor independent. We also accounted for marital status and employment status, which were not considered in previous papers, but may confound the association between hearing loss and physical activity. Moreover, our findings of a positive association between hearing and physical activity in the range of normal hearing (BPTA <20 dB HL) but a negative association in the range of mild to more severe loss (BPTA ≥20 dB HL) is novel. One of the mechanisms that may explain the association between hearing loss and lower physical activity is that hearing loss may lead to social isolation29 and reduced life space mobility,30 which limit an individual’s opportunities to be physically active.31 This mechanism, however, would only be true for those adults for whom the degree of hearing loss is high enough to affect their social life, which is supported by our findings from the linear spline regressions. Another mechanism that may explain the associations we observed is the reduced perception of environmental cues that help with spatial calibration when moving among those with hearing loss.32 The nonlinearity that we observed in the association between hearing loss and daily movement has also been described for the association with depressive symptoms and cognition.34 While the previous literature in the hearing loss field has used the 25-dB HL threshold to define mild hearing loss, there is a lack of consensus on what BPTA should be used to define hearing loss. In the most recent world report on hearing from the WHO, a threshold of 20 dB HL is used.27 Moreover, even lower thresholds have been proposed, such as 15 dB HL.35Our findings agree with those of Kuo et al,21 Therefore, our findings cannot be related to the physical activity recommendations for Americans (≥150 minutes/week of moderate activity or ≥75 minutes/week of vigorous activity), which are widely used for public health messaging. Fourth, our sample size for the group with moderate or greater hearing loss was small (n = 28), which may have limited our statistical power to detect differences in physical activity levels across hearing loss groups. Finally, given the small number of participants who reported hearing aid use, we decided to exclude them from our analysis; thus, we were unable to assess whether hearing aid users were more active than nonusers.Our study has some limitations. First, this is a cross-sectional analysis; thus, temporality cannot be ascertained. Second, during the 2011-2012 NHANES cycle, adults older than 69 years were excluded from audiometric testing; therefore, we may not be observing many NHANES participants in whom hearing loss may be more prevalent and severe. Additional studies confirming these associations in older populations are warranted. Third, there are currently no minute-level MIMS units thresholds to define physical activity intensities.Despite these limitations, our study has several strengths. As additional devices to objectively measure physical activity are implemented in research studies, a comparison of results across studies becomes more challenging since many studies have used device-specific algorithms to summarize accelerometry data. Our study incorporated the use of MIMS units, which use a nonproprietary open-source and device-independent algorithm to objectively derive movement from raw accelerometry data. Thus, MIMS units may help with harmonization efforts across studies. We conducted a series of sensitivity analyses, including moving the knot of our linear spline to evaluate different thresholds at which we observed the association between hearing and physical activity. All analyses suggest that adults with hearing loss were less active; however, only the analysis with a linear spline were statistically significant.37Our study supports the hypothesis that hearing loss is associated with lower levels of physical activity, characterized using novel MIMS units metrics. Reductions in physical activity as a result of hearing loss may be an important mechanism contributing to the association between hearing loss and adverse health outcomes but will require further investigation in longitudinal studies. Furthermore, whether hearing rehabilitation strategies may help attenuate the adverse consequences of hearing loss on physical activity and other downstream outcomes requires examination in future studies.


Tricholoma matsutake, is a hotspot of microbial habitation and interaction. However, microbial diversity within this microniche structure of the EMF is rarely investigated. Furthermore, there is limited information concerning microbiomes associated with sporomes belonging to the ubiquitous fungal phylum Basidiomycota, particularly with respect to fungus-EMF interactions. In this study, we conducted high throughput sequencing, using ITS  and 16S rRNA  marker genes to characterize and compare fruiting body microbiomes in the outer (pileipellis and stipitipellis) and inner layers  of the fruiting body of T. matsutake. Our results show the number of unique bacterial operational taxonomic units (OTUs) among the different compartments ranged from 410 to 499 and was more than double that of the shared/common OTUs (235). Micrococcales, Bacillales, Caulobacter, and Sphingomonas were the primary significant bacterial taxa within the different compartments of the dissected T. matsutake fruiting body. Non-parametric multivariate analysis of variance showed significant compartmental differences for both the bacterial and the fungal community structure within the T. matsutake fruiting body. The metabolic profiling revealed putative metabolisms  and the biosynthesis of secondary metabolites to be highly enriched in outer layers; in the inner parts, the metabolisms of energy, cofactors, vitamins, and lipids were significantly higher. This study demonstrates for the first time the distinct compartmentalization of microbial communities and potential metabolic function profiles in the fruiting body of an economically important EMF T. matsutake.The uniquely compartmentalized fruiting body structure of the ectomycorrhizal fungus (EMF)  The fungal phylum Basidiomycota is one of the most diverse fungal groups, with a presence over a wide environmental range . Nearly Bradyrhizobium, Roseiarcus, and Pseudolabrys during primodium formation and fruiting body development of Phlebopus portentosus   reductase (K00059), and a LacI family transcriptional regulator (K02529) /glutamyl-tRNA (Gln) amidotransferase subunit A (K02433) and a cold shock protein  (K03704), while in the outer layer of T. matsutake, multiple sugar transport system permease proteins (K02025 and K02026) were a strongly increased functional type (The KEGG ortholog homologous function predictions in (K02529) . In the nal type .T. matsutake sequences , 319 fungal OTUs remained. Among these 319 non-host fungal OTUs, 219 fungal OTUs (68%) were assigned to specific trophic modes. Out of these functionally assigned OTUs, 111 OTUs with the “possible” confidence ranking were removed, and the remaining 40 and 68 OTUs that belonged to the acceptable confidence rankings assigned to the “highly probable” and “probable” types were used for downstream functional analysis.In the fungal ITS sequence analyses, after filtering out the Although the (within column) numbers of fungal OTUs were different, the relative abundance of fungal trophic modes was similar . The funT. matsutake fruiting body. Generally, more microbial species tended to inhabit the inner layer of T. matsutake as compared to the outer layer. This trend of higher microbial diversity at the inner layer was also found for another EMF, Thelephora ganbajun  [In support of our first hypothesis 1 (H1), both bacteria and fungi selectively inhabited different compartments of the omycota) . Additionally, a more complex microbial partitioning was observed for the studied EMF within the micro-niches of the inner layers, and these niches include parts such as the stipe, pileum, and the lamellae. We expect this phenomenon is associated with microbial functioning. For instance, the pileum might be responsible for the development of adjoining compartments (lamellae and pileipellis), and may exhibit higher nutrient availability due to the associated growth-promoting bacteria . T. ganbajun, the thick interior context of the fungal fruiting body was found to have a greater abundance of C- and N-fixing genes, and sourcing energy and nutrition to the associated microbes [T. matsutake may function as (i) an important reservoir of nutrients and (ii) a microbial hotspot niche as indicated by its highest microbial richness, in comparison to the other compartments of the fruiting body.In a recent investigation by Liu et al. (2021), while studying microbial functional gene compartmentalization in the EMF microbes . SimilarT. matsutake fungal body, and may have greater biological relevance by assisting in secondary metabolite production [T. matsutake could be enriched by—and enrich—N-fixing bacteria, as revealed by the much higher relative abundance of Moraxellaceae  in this micro-niche. The unique numbers of endofungal bacteria were much higher than those of the endofungal fungi. This higher prevalence of endofungal bacteria within the fruiting body of T. matsutake further elaborates their critical role in fruiting body differentiation/compartmentalization and is in line with the earlier study [T. matsutake compartments (H1). In particular, Bacteroidetes predominated in the outer layer of this EMF, while Proteobacteria were mainly localized in the inner layer. This highlights the role of Proteobacteria occupying micro-niches for nutrient acquisition and is in line with their copiotrophic life strategy [T. matsutake nutritional components  was not included in our work, we observed significantly higher potential metabolisms involving vitamins, lipid, and amino acid synthesis in the inner layer of T. matsutake fruiting bodies in comparison to the compartments. Our results clearly show . For bacteria, there were 499, 491, 450, 441, and 410 OTUs found exclusively in the pileum context, stipitipellis, stipe context, pileipellis, and lamellae, respectively. Meanwhile, for fungi there were 205, 52, 52, 47 and 33 exclusive OTUs for stipe context, pileum context, pileipellis, stipitipellis and lamellae, respectively. This shows a clear microbial compartmentalization in the studied maturation stage, which may be related to the biochemical, environmental and nutritional differential conditions in the evaluated microniches, i.e., changes in pH, CO2 concentration, volatile organic compounds, key odorants or nutritional composition [The context of the stipe also plays significant roles in providing structural support to the fruiting body skeleton as well as being a site of secondary metabolite production . The quaoduction ,30. Furter study . Microbistrategy ,32. Suchstrategy . Althougrly show  that theposition ,35. Howeposition . Additioposition ,38, the T. matsutake compartments. Micro-niche partitioning disparity of the T. matsutake individual compartments (outer vs. inner layer), which assembles unique microbiomes, may explain this difference. Similar spatial niche-induced microbial recruitment and assembling trends were reported previously among fungus-associated components [T. matsutake mushrooms should be more considered as the host, with the endophytic and epiphytic microbes as either commensals and/or parasites that rely on the T. matsutake mushrooms to provide nutrients. Direct evidence for this is lacking, but preliminary potential functional profiles (using PICRUSt) support the idea of T. matsutake as a nutritional repository for endofungal microorganisms. Amino acid metabolism was the strongest identified function within the fruiting bodies of T. matsutake. As amino acids are the building blocks of proteins, polypeptides, and other nitrogenous biomolecules, their exchange between T. matsutake and its associated microbes could be of great importance. In agreement with our second hypothesis (H2), microbial potential metabolic functions varied among the mponents ,40 and wmponents ,41. SuchT. matsutake fruiting bodies (using 90% ethanol) possess moderate anti-bacterial properties against Escherichia coli , Staphylococcus aureus (38%), and Salmonella enterica (34%) [At the bacterial family classification level, we found that the Pseudomonadaceae and Sphingobacteriaceae showed higher relative abundances in the outer layer of the fruiting body. This could indicate their ecological roles in secreting antibiotics and substance-degrading enzymes such as hydrolases, chitinase, lysozyme, and xylanase ,43. Therca (34%) . Tricholoma matsutake fruiting bodies showed that the main endofungal fungi trophic mode was the symbiotroph  [Thelephora, Tuber and Tomentella genera. As these are also common EMF with strong infectivity, they could perform similar functions such as promoting phosphorus absorption and host growth as they do for host trees. Certain bacteria  were also differently presented in T. matsutake fruiting bodies, known as saprotrophic decomposers by secreting chitinases [Neofusicoccum, Mycosphaerella, Ascochyta, Podosphaera, Amphobotrys, Phaeoacremonium, Trichothecium, Cylindrocarpon, Gibberella, Slopeiomyces, Libertella, Monographella, Waitea, Anthracoidea, and Rhizophydium [Pseudomonas species  [P. putida was found in all T. matsutake compartments, with preference in the stipe context more than other parts  ,46, withitinases ,19. Compitinases , wood saitinases , and dunitinases . In the itinases . For end putida) ,52. In ter parts . EndofunT. matsutake work is a seminal investigation whose strength is to show the diversity and specificity of microbiomes in different compartments. However, we have to recognize that the functional relevance and contribution of the microorganisms associated with T. matsutake fruiting bodies require deeper analysis with more powerful tools in the future.To our knowledge, this is the first study of microbiome partitioning in fruiting bodies in an Agaricales fungus. The Tricholoma matsutake, we demonstrated that EMF do not merely function as fruiting bodies for spore dispersal, but also localize different microbial communities within their structures to generate microbial hotspots of varying importance and complexity. We have shown for the first time that in an Agaricales fruiting body, the fungus-related microbial environment is more complex than previously expected, in the context of not only the localized microbial taxa, but also based on their potential metabolic functions as well. Each compartment of the EMF fruiting body can be treated as a unique site of diverse secondary metabolite production, encompassing microbial hotspots with greater metabolic potential. Unlike the conventional macroscopic classification of environmental fungi, we explored an advanced approach of segregating the fungal fruiting body into its component parts, based on the associated microbes residing in each compartment. Such compartmentalization and associated metabolic functions therein can be explored more in light of the state-of-the-art techniques such as multi-omic  approaches in future studies. As an important component of the terrestrial environmental, ectomycorrhizal fungi play vital roles in biogeochemical nutrient cycling in forest ecosystems. Based on our study of a selected model EMF,


However, for people with cognitive impairment, factors in addition to the PTA can play a role in their self-reported difficulties. Patients with cognitive impairment may experience more hearing difficulties due to their brain processing sounds abnormally, irrespective of PTA. (2) Methods: Three groups of hearing aid users who had normal cognition, mild cognitive impairment and dementia were recruited. Self-reported hearing abilities were assessed with the modified Amsterdam inventory for auditory disability (mAIAD) and the speech, spatial and qualities of hearing scale (SSQ). (3) Results: The SSQ and mAIAD scores were highly correlated with each other for all three groups. However, a correlation with objective PTA was found in the normal cognition but not the cognitively impaired groups. Self-reported hearing scores were associated with cognitive scores for the dementia group (4) Discussion: In people with combined cognitive and hearing impairment, PTA alone may be a poor predictor of hearing abilities. Subjective hearing questionnaires together with hearing tests may provide a better understanding of their hearing difficulties. Patient-reported outcomes have been used in clinical trials to capture what matters most according to the daily life experience of people living with dementia (PLWD). Traditionally, the validity of self-reported hearing disability measures has been assessed using their correlations with the pure-tone average (PTA) hearing loss. These studies have all been conducted using non-cognitively impaired adults with and without hearing impairment ,2,3. HowAmong older adults, an association was found between (self-report) hearing handicap and cognitive function independent of the PTA . PatientIn this paper, we explore hearing handicap using two commonly used self-report hearing questionnaires among older adult hearing aid users with normal cognition versus those with mild cognitive impairment and those with dementia. The study aimed to investigate the interpretation of self-report hearing questionnaire scores for people with differing cognitive status. We assessed validity in terms of the questionnaires’ correlation with each other  and with the PTA . In addition, to understand the relationship between cognitive impairment and objective and subjective hearing tests, the correlations between the subjective (self-report)/objective (PTA) hearing tests and the cognitive tests were explored.The project was approved by the UK National Health Services (NHS) Ethical Committee IRAS247176, under the University College London Joint Research Office (JRO) sponsorship ID 18/0306. The study protocol was registered in clinicaltrial.gov (NCT03648502).The inclusion criteria were age ≥65 years with documented hearing loss . The exclusion criteria for all groups were uncorrected visual impairment, physical disability(s) that might impair performance on the written/drawing elements of the tests, and congenital/childhood-onset hearing loss (<18 years old age), as reported by participants.Thirty adults were recruited via recruitment flyers and posters distributed in the hearing aid centre at the Royal National Throat Nose Ear Hospital (RNTNEH), London, United Kingdom. To ensure normal cognition, only those with a General Practitioner’s Assessment of Cognition (GPCOG) score = 9 or GPCOG score = 5–8 with informant/carer interview score = 4–6 (no memory concern for the carer) were recruited .Thirty adults diagnosed with mild cognitive impairment (MCI-HI) and 15 with dementia (D-HI) were recruited via clinician referral and research registry in the memory clinics at Camden and Islington NHS Foundation Trust, London, United Kingdom. The diagnosis of MCI and dementia were based on ICD-10 criteria .We selected two questionnaires that are commonly used for assessing hearing difficulty among hearing aid users. The modified Amsterdam inventory for auditory disability (mAIAD)mAIAD consists of 28 questions that are divided into 5 domains (Speech) Intelligibility in noise(Speech) Intelligibility in quietAuditory localisationRecognition of soundDiscrimination of sound.The responses are scored on an ordinal scale ranging from 0 indicating the most difficulty in hearing to 3 indicating the least difficulty, and as ‘almost never’ (scored 0), ‘occasionally’ (scored 1), ‘frequently’ (scored 2) and ‘almost always’ (scored 3). The questionnaire was validated using an adult non-cognitively impaired cohort with varying hearing abilities. The test has good internal consistency and high test–retest reliability along with high validity The speech, spatial and qualities of hearing scale (SSQ)The questionnaire was validated in outpatient clinics to evaluate hearing difficulties . The resThe score for each domain and the overall scores of both questionnaires were calculated by averaging the responses in each domain and all questionnaire items respectively . Audiograms were conducted for every participant, according to the British Society of Audiology protocol  during twww.mocatest.org (accessed on 17 June 2021).A visual adaptation of the MoCA 8.3 adapted for people with hearing impairment  was usedSPSS version 25 was used. The baseline characteristics  of each group were compared with ANOVA. Pearson correlation was used to evaluate the questionnaires and PTA correlations, having checked test assumptions including the data with a normal distribution. A Pearson correlation coefficient ranging from 0.1 to 0.3 (or −0.1 to −0.3) was considered a weak correlation. A correlation ranging from 0.3–0.5 (or −0.3 to −0.5) was considered a moderate correlation. A correlation of more than 0.5 (or less than −0.5) was considered a strong correlation .This is an initial exploratory study aimed at hypothesis generation. The Bonferroni correction for multiple comparisons were therefore not performed as it would increase the probability of false negatives in a manner too stringent for an exploratory study.There was no significant difference in the subjective hearing assessment and PTA, defined by the scores of the hearing questionnaires and the better ear PTA, respectively, among the three groups as shown in P < 0.001). The strongest correlations were observed between the overall scores for the two questionnaires and between the equivalent domain subscores for each questionnaire as shown in Moderate to strong correlations were found for most domains between the overall mAIAD and SSQ scores, and the PTA for the NC-HI group as shown in For the NC-HI and MCI-HI groups, the correlations of MoCA score and hearing ability (objective and subjective) were weak and not significant as shown in While the questionnaire scores were correlated with the PTA value for the NC-HI group, this was not the case for the MCI-HI and the D-HI groups, where the correlations were weaker and mostly not statistically significant. However, despite there being no correlation with PTA, the self-reported hearing problems were associated with cognitive scores for the D-HI group.The mAIAD scores for the spatial and sound localisation domains of the questionnaires were not correlated with PTA even for the NC-HI group. Sound localisation hearing difficulties are commonly found in cognitively impaired groups ,17. ThisPrevious research has shown that the mAIAD and SSQ questionnaires could identify auditory difficulties in a cognitively normal group with normal PTAs when they had a clinical diagnosis of auditory processing disorder . The demThe two questionnaires were positively correlated for groups of all cognitive abilities. This suggested that the self-report questionnaires reflected the hearing handicap of the participants regardless of their cognitive status. Previous research found that self-reported hearing difficulties among patients in a memory clinic were often less than those reported by their caregivers and expected from their PTA . HoweverThe relationship between the self-reported hearing difficulties (mAIAD) score and cognition (MoCA score) was significant for the dementia group but not for the mild cognitive impairment and the normal cognition hearing-impaired group. For the dementia group, the greater cognitive impairment they had, the more they reported hearing difficulties. As discussed earlier, this association may not necessarily be a result of “peripheral” type hearing loss as reflected by the PTA. This finding agrees with research among Asian older adults  showing Subjective hearing difficulties are not valid as proxies for PTA in subjects with dementia. Combining subjective hearing difficulties from the questionnaires (reflecting their auditory processing ability) with the PTA  should be promoted for the cognitively impaired group.This research explored the relationship between hearing and cognition in groups with combined cognitive and hearing impairment. However, due to the small sample size, the lack of a significant correlation for some groups particularly the dementia group could be due to a lack of power rather than the absence of an effect.This study was exploratory and did not incorporate the correction of the significance levels to allow for multiple comparisons. Replication of findings in a larger cohort is required to confirm the study findings.Scores for the self-report questionnaires among cognitively impaired older adults may be a predictor of their hearing difficulties. Self-report questionnaires in assessing peripheral hearing difficulties assessed by PTA may not be valid in this group since the correlation of the questionnaire results with the objective hearing test is weaker than in those with normal cognition. In the dementia hearing-impaired group, a correlation was observed between scores for the MoCA cognitive assessment scores and their self-reported hearing difficulties, but not with the PTA.In people who have a combined cognitive and hearing impairment, subjective hearing assessments with questionnaires and more extensive audiometric tests may have a role in supplementing the PTA in facilitating the overall understanding of the hearing difficulties.


N-glycosylase (EC 3.2.2.22) by hydrolyzing and depurinating A-4324 in α-sarcin/ricin loop on 28S rRNA of rat ribosome. Its interaction with acidic ribosomal stalk proteins was revealed in 2007, and its trafficking in mammalian cells was elucidated in the 2000s. The adverse drug reactions, such as inducing immune responses, short plasma half-life, and non-specificity, somehow became the obstacles to its usage. Immunotoxins, sequence modification, or coupling with polyethylene glycerol and dextran were developed to improve the pharmacological properties. TCS has nicely shown the scientific basis of traditional Chinese medicine and how its research and development have expanded the knowledge and applications of ribosome-inactivating proteins.Tian Hua Fen, a herbal powder extract that contains trichosanthin (TCS), was used as an abortifacient in traditional Chinese medicine. In 1972, TCS was purified to alleviate the side effects. Because of its clinical applications, TCS became one of the most active research areas in the 1960s to the 1980s in China. These include obtaining the sequence information in the 1980s and the crystal structure in 1995. The replication block of TCS on human immunodeficiency virus in lymphocytes and macrophages was found in 1989 and started a new chapter of its development. Clinical studies were subsequently conducted. TCS was also found to have the potential for gastric and colorectal cancer treatment. Studies on its mechanism showed TCS acts as an rRNA  Trichosanthes kirilowii (Gua Lou) from the family of Cucurbitaceae [Qianjin Yifang by Sun Simiao was the first recording of Tian Hua Fen in the 7th century. The others, including Taiping Royal Prescriptions by Wang Huaiyin and Compendium of Materia Medica by Li Shizhen have also recorded it in prescriptions to deal with abortion, abnormal menstruation, and retained placenta [Trichosanthin (TCS) is generated from the extract of the root tuber of bitaceae . The roobitaceae . The traplacenta .Chongxiu Zhenghe Jingshi Zhenglei Beiyong Bencao show two Gua Lou from Hengzhou and Junzhou [T. cucumeroides (Ser.) Maxim (Wang Gua). Others such as T. cavaleriei Levl. (Chang Mao Gua) and T. hupehensis. C. Y. Cheng et C. Y. Yueh (Hu Bei Gua Lou) have more side effects such as nausea and vomiting. Gua Lou that produces TCS mainly comes from Boxian in Anhui, Anyang in Henan, Pudong in Shanghai, and Nantong in Jiangsu [Ancient records show different plant resources of Tian Hua Fen. Records in  Junzhou . The lat Junzhou ,6. Some  Jiangsu  , Asarum sieboldii (Xi Xin), the root of Euphorbia fischeriana (Lang Du) or with three more herbs, including Angelica dahurica (Bai Zhi), the root of Kaempferia galanga L. (Shan Nai), and the root of Nardostachys chinensis Batal (Gan Song).Because of the lack of western drugs, Tian Hua Fen was used extensively in China for inducing abortion in the 1960s . The preThe herbs were crushed to make a liquid extract as a medicated vaginal suppository (4) to induce abortion. However, because of severe adverse drug reactions (ADR) such as fever, tetter, and abdominal pain, studies on revising the prescription were carried out. In 1965, researchers in Wuhan and Nanjing, China, conducted thorough studies on the chemical, pharmacology, and clinical characteristics of the prescriptions to improve its pharmacological performance. The original prescriptions were simplified to contain only Tian Hua Fen and Ya Zao, called “Tian Zao He Ji” in powder form as capsules in the late 1960s ,9,10. TiHowever, ADR was still a concern in its application. In 1966, researchers in Shanghai further isolated and purified components in Ya Zao and Tian Hua Fen. One death report in clinical application in 1969 alerted researchers again on the impact of ADR. The researchers showed that only Tian Hua Fen could induce abortion in pregnant mice, while Ya Zao only causes muscle festering. This suggests Tian Hua Fen was the component for abortifacient, while Ya Zao was only an enhancer, profiting from its surfactant characteristic ,11,12.Trichosanthes Kirilowii in different ways are shown in Tian Hua Fen itself was precipitated and made as injections, first used in 1972. The dosage was 5 mg per dose. It was then developed as more purified injections in 1975 with only 2 mg per dose. In 1982, the precipitated powder was further crystallized in barbitone buffer and came out as the single protein powder of trichosanthin (TCS) . The dosThe clinical application was synergistically developed with its mechanism study. The mechanism on its midterm abortion and anti-early pregnancy effects were reported relying on the damage of syncytiotrophoblast cells  and the The clinical studies of Tian Hua Fen and crystallized TCS powder were firstly on midterm abortion. They then expanded to anti-early pregnancy, ectopic pregnancy, molar pregnancy, and chorionic epithelioma. Tian Hua Fen was a topical medication for the vagina when used as a prescription for TCM. Then it was used for muscle, amniotic cavity, cervix uteri, or intrauterine injections with a simplified prescription and further purification .For midterm abortion, gravidas in 12–24 weeks pregnancy were treated with injections after a skin test . To avoiTian Hua Fen was also used in stopping early pregnancy . As withThe primary structure (protein sequence) of TCS was first reported in the Symposium on the Organic Chemistry of Medicinal Natural Products held by the International Union of Pure and Applied Chemistry (IUPAC) in Shanghai in 1985 . Wang etT. kirilowii, and cloned and sequenced the cDNA of TCS [E. coli [Our group prepared the cDNA library of the root tuber of A of TCS . Recombi[E. coli . This opN-glycosylase (EC 3.2.2.22) in 1992 and was classified as a ribosome-inactivating protein (RIP) [N-glycosidic bond of A-4324 on the conserved sarcin–ricin loop (SRL) on the 28S rRNA of rat ribosome [Trichosanthin was shown to be an rRNA in (RIP) . As within (RIP) , TCS catribosome .E. coli but can react on naked 23S rRNA. Sequences of the 28S and 23S rRNA in α-SRL are highly conserved. Endo et al. showed that ricin can retain its specificity mimic α-SRL on 35-mer synthetic oligoribonucleotide [RIPs have substrate specificity. Taking the ricin A chain (RTA) as an example, RTA cannot depurinate intact ribosomes from cleotide , which cDepurination of the SRL inhibited the binding of the elongation factors to the ribosomes and suppressed the activation of GTPase ,42,43. IIn the 1990s, crystal structures of TCS and other RIPs together with their substrates or analogues, including Formycin 5′-monophosphate (FMP), adenyl(3’→5’)guanosine (ApG), ATP, adenosine (ADO), cytine monophosphate (CMP), tubercidin (Tub), and NADPH, were studied ,46,47,48The structure of TCS a consistN-glycosylase is contributed from the subsequent nucleophilic attack of C-1 of the water molecule to the ribose.In the proposed mechanism ,36,37, tTCS interacts with ribosomal stalk P-proteins and L10a ,58. RiboIn 2009, the co-crystallization of this C-terminal 11 amino acids peptide (C11-P) and TCS revealed the binding mode c. The riIn 1989, McGrath et al. first reported the inhibitory activity of TCS on HIV replication in HIV-sensitive T-lymphoblastoid cells (VB cell line) ,73. HighN-glycosylase activity and enhancement of chemokines to stimulate chemotaxis and activation of pertussis toxin-sensitive G protein were considered the most important [The mechanism of the anti-HIV activity of TCS is not entirely clear. However, its rRNA mportant . In recemportant .Temporary interaction of TCS with HIV-1 long-terminal repeats (LTRs) can affect the HIV integration step . TCS-enrLendray et al. in 1991 reported the treatment of GLQ223 on Simian immunodeficiency virus (SIV)-infected rhesus macaques [In the 1990s, after McGrath et al. reported the anti-HIV potential of TCS, the Food and Drug Administration (FDA) permitted the clinical study of TCS on HIV using purified and formulated preparation GLQ223 ,83. UnofFor the Phase I clinical study, increasing dosages of TCS were used to measure the kinetic parameters such as biological half-life time (t1/2), serum clearance rate, distribution, and ADR observation ,20,23,83Phase II clinical trials were conducted in azidothymidine (AZT)-invalid HIV patients ,85,86,87The clinical studies of TCS are listed in Trafficking of RIPs into the cells and interacting with ribosomes are essential steps for their cytotoxicity . TCS is Without the assistance of the lectin B chain, TCS uses receptor-mediated endocytosis and non-specific entry to enter the cells . In 2003TCS has potential anti-cancer activities on tumors in the female reproductive system , immune system, digestive system , blood system, respiratory system, and others . The staIn the SGC-7901-based gastric cancer transplant tumor nude mouse model, TCS also suppressed tumor growth in vivo with an effective suppression dose of 0.5 mg/kg, far lower than the median lethal dose (LD50) 13.4 mg/kg. Combined administration of TCS with fluorouracil (5-FU), mitomycin (MMC), and interferon α-2b showed higher cytotoxicity on gastric and colorectal cancer cell lines. Takemoto et al. in 1998 reported the anti-tumor activity of the N-terminal sequence of trichosanthin in vivo [The use of TCS to combat cancer is an active research area. Chen et al. showed that TCS inhibits the proliferation, migration, and epithelial–mesenchymal transition of human cervical cancer cells. These were considered mediated by inhibiting the STAT5/C-myc signaling pathway . Smac waThe engineering and modification studies of RIPs include these aspects: (a) immunotoxins (ITs) made to improve pharmacological performance; (b) engineering of signal sequences to improve specificity; (c) coupling with PEG or dextran to increase the plasma half-life. A pharmacokinetic study of TCS in mice showed that the kidney excreted TCS through urine; the half-life was 8.4–12.7 min. Ko et al. reported coupling with dextran could prolong plasma half-life and minimize renal loss . TCS couOur group also tried to improve the specificity of RIPs by linking RIPs with HIV-1 protease recognition sequences to the C-termini or the internal inactivation region of Maize RIP variants or RTA ,111,112.Immunotoxins usually consist of a vector part for interaction and a toxin part as the “bullet”. The vectors need high affinity, specificity, high stability, and long half-life to the target. Clusters of differentiation (CD) on hematological cells’ surfaces are among the most popular used ITs targets. Monoclonal antibodies (mAb) against various cancer antigens, hormones and growth factors, and cytokines were also used ,107. TheT. Kirilowii, and has high homology with TCS [In 1987, Wang et al. in China reported using TCS conjugated with monoclonal antibody (mAb) on liver cancer . In 1988with TCS . A cleavwith TCS . ITs baswith TCS . These sIn the 1990s, researchers reported the use of ITs of TCS with Hepama-1 (human hepatoma) , CMU15A Various historic medicines are still sparkling with the modification of advanced technology. Insulin was isolated from the internal secretion of the pancreas by Banting and Macleod in the 1920s . It was N-glycosylase (EC 3.2.2.22) supported its engineering for improvement. These made TCS become a showcase of bench-to-bedside translation and drug development from TCM.For the clinical applications of TCS, there are three active areas of work. Before the 1980s, it was abortion. In recent decades, anti-HIV and anti-cancer have become more active. Clinical requirements first motivated the application of TCS as an abortifacient. The ADRs reflected from the clinical application then drove the modification and engineering of TCS. New biological activities inspired novel applications. Its molecular and structural basis as rRNA Nicotiana tabacum was inhibited by exogenous application of recombinant TCS. It also caused a delay in the development of mosaic symptoms by TuMV in Brassica parachinensis [As well as clinical applications, TCS has been shown to inhibit plant viruses. Local lesion formation by turnip mosaic virus (TuMV) in leaves of hinensis . The trahinensis ,133.Lyophyllum shimeji. This RIP is a member of the peptidase M35 superfamily but has potent rRNA N-glycosylase activity [After TCS, various other RIPs were studied. The distribution of RIPs was also expanded from plants to bacteria , animalsactivity .For pharmaceutical uses, immunogenicity, short plasma half-life, and non-selective cytotoxicity were the major issues to be overcome. Its immunogenicity may cause reduced efficacy through chronic exposure. As well as coupling with polyethylene glycerol and dextran, deimmunization by depleting T-cell epitopes on the protein can be a good way to reduce immunogenicity in designing ITs . Recent There are still many opportunities in the research and development of TCS. New techniques in nanoparticles, co-delivery systems, and tumor-targeting protein delivery systems ,145 may In conclusion, TCS has emerged from an abortifacient to an anti-HIV and anti-cancer agent. Over the years, modifications have been carried out to improve pharmacological performance, and mechanism studies have been conducted to understand the enzymatic reaction. TCS has become a showcase for the mechanism of function and applied values of this class of proteins. The research and development on TCS have also revealed the hidden treasure in Chinese medicine.


Beauveria bassiana does not need to rely on insect hosts to complete its life cycle. The diverse lifestyles of this fungus, saprophyte, pathogen, and symbiont, provide a unique system, with available genetic tools, to examine host-pathogen interactions, plant-fungus mutualistic relationships, and fungal development. This commentary highlights overlooked pathogenic and mutualistic aspects of B. bassiana that assist this fungus in shifting along the saprobe/parasite/mutualist continuum. Addressing these knowledge gaps and scrutinizing valuable players driving such a spectrum of ecological interactions will enrich our knowledge of fundamental environmental microbiology and help develop new approaches to pest control and sustainable farming.Fungal pathogenicity toward insects has independently evolved several times, resulting in specialist and generalist pathogens, some of whom have maintained aspects of their previous lifestyles. Being able to grow as an endophyte  or saprophyte (recycling nutrients back into the environment), the generalist (broad-host-range) fungus  Beauveria bassiana can infect more than 700 insect species from all the major insect orders  examine the drivers of fungal evolution, distribution, parasitism, and mutualism and (ii) develop novel approaches for sustainable pest control and farming.Entomopathogenic fungi are significant factors in natural insect epizootic infections worldwide. Particularly, t orders . Like ot cadaver , 3  toxins. While lipid assimilation/detoxification/hydrolysis is known to contribute to the ability of B. bassiana to overcome the antimicrobial barrier of the insect cuticle , the production of toxins may mediate (i) transitions during host infection and death , (ii) transitions during rhizosphere interactions and endophyte establishment and persistence, and (iii) antagonism toward microbial competitors. Understanding the mutualistic lifestyle of B. bassiana remains a nascent field in the development of hypotheses on plant-fungus interactions, including resistance to biotic/abiotic stresses and plant growth promotion. Our laboratory currently examines the molecular mechanisms underlying B. bassiana endophytic growth and B. bassiana-mediated plant resistance toward insects to gain a fundamental understanding of these processes and to exploit B. bassiana applications better.Although significant progress has been made in our understanding of the fundamental and applied aspects of this fungus, this commentary addresses facets of ontinuum . These iB. bassiana needs to adhere to the insect epicuticle, a hydrophobic layer consisting of lipids and other compounds, some of which have potent antimicrobial activities . Much of the success of B. bassiana as an insect pathogen relies in its ability to overcome this antimicrobial barrier. B. bassiana contains a subset of cytochrome P450 monooxygenases which allows it to use  lipids as a sole source of carbon, generating fatty acyl coenzyme As (acyl-CoAs) for membranes, lipid storage, or β-oxidation (B. bassiana (and other pathogenic fungi) increases lipid droplet formation and lipid mobilization, which have been linked to virulence  and to greater stress tolerance and spore dispersal . One limitation to pursuing this line of research is the presence of multiple lipases  with poAfter breaching the insect cuticle and reaching the hemocoel, the fungus undergoes a dimorphic transition to grow as yeast-like cells, termed hyphal bodies . These cB. bassiana bacterial toxin-like (putative) toxins, including seven Cry protein-like toxins, 13 heat-labile enterotoxins, and three zeta toxins, have been identified in genomic analyses  of infection via the gut after the ingestion of fungal spores. Alternatively, zeta toxins may not have direct effects in fungal infection and could act as bactericidal agents during fungal necrotrophic/saprophytic growth, similar to what has been reported for the B. bassiana secondary metabolite oosporein  pathway. Plant SA alters the composition and assembly of the plant microbiome, and plants expressing constitutive production of SA show slower fungal colonization  is needed and will provide critical information on B. bassiana growth dynamics within the host plants. Furthermore, like other fungal endophytes, B. bassiana has the potential to bypass the activation of plant SA-dependent systemic acquired resistance by producing a  salicylate hydrolase. This enzyme potentially would enable the fungus to grow endophytically without causing drastic changes in plant immunity.Knowledge on the molecular basis mediating nization , 12. LowB. bassiana are (i) any impacts on arthropod pests feeding on colonized plants, (ii) the extent to which B. bassiana may safeguard plants from plant pathogens, and (iii) the degree and conditions driving growth promotion and increased plant abiotic stress tolerances  and/or indirectly by deterring arthropods from ovipositing on or colonizing the plant. B. bassiana can be found as a natural epiphyte on many plants, which supports potential direct parasitism via topical acquisition of fungal spores from the plant canopy. Alternatively, Cry-like toxins and enterotoxins could be produced in planta during fungal endophytic growth, providing plants with direct systemic protection against insect pests, an idea we are currently exploring.Unique and understudied aspects of endophytic colonization by lerances . While sB. bassiana and/or B. bassiana-induced plant VOCs attract or deter arthropod colonization. Potentially, these VOCs may enable herbivore and nonhost signaling . By attracting insects, VOCs could benefit the fungus by providing hosts and potentially even benefit the plant via nitrogen transfer from fungus-mediated insect mortality (One of the major gaps in our understanding of the fungus-plant-insect interaction is the role(s) of communication signals arbitrated by volatile organic compounds (VOCs). Fungal endophytes can release VOCs causing major changes in the plant chemistry. However, it is not clear if ortality , or by rB. bassiana parasitic and mutualistic interactions requires a more complete insight into the cross talk between B. bassiana, insect host, and plant partners. In this regard, combined “omics” analyses  of fungus-challenged insects and plants can provide simultaneous profiles of the nature of these interactions. Plants with low genetic redundancy, e.g., liverworts, could represent simpler models than other land plant systems to dissect the molecular basis of the fungus-plant interaction. Cell biological (determining where in the plant) and genetic (gene knockout) approaches can provide more nuanced understanding of the mechanistic bases underpinning fungal infection and mutualism. Finally, knowledge gain from these studies can be translated into important applications impacting food safety and security, biodiversity, and crop yield and resilience.Identifying the main participants in


Here, we discover that Au1/2Ag1/2CN forms diamond-shaped nanocrystals epitaxially grown on pristine graphene surfaces. The nanocrystals synthesized by a simple drop-casting method are crystallographically aligned to lattice structures of the underlying graphene. Our experimental investigations on 3D structures and the synthesis conditions of the nanocrystals imply that the rhombic 2D geometries originate from different growth rates depending on orientations along and perpendicular to 1D molecular chains of Au1/2Ag1/2CN. We also perform in situ TEM observations showing that Au1/2Ag1/2CN nanocrystals are decomposed to Au and Ag alloy nanocrystals under electron beam irradiation. Our experimental results provide an additional example of 1D cyanide chain families that form ordered nanocrystals epitaxially aligned on 2D materials, and reveal basic physical characteristics of this rarely investigated nanomaterial.Epitaxial synthesis of inorganic nanomaterials on pristine 2D materials is of interest in the development of nanostructured devices and nanocomposite materials, but is quite difficult because pristine surfaces of 2D materials are chemically inert. Previous studies found a few exceptions including AuCN, AgCN, CuCN, and Cu Epitaxial synthesis of inorganic nanomaterials on pristine surfaces of 2D materials ,2,3,4,5 0.5Au0.5CN are epitaxially aligned onto various 2D materials including graphene, h-BN, and MoS2. One-dimensional (1D) molecular chain structures, which are a characteristic feature of these metal cyanides, are responsible for their preferential growth and epitaxial alignment on 2D materials, because the 1D chain structures would enable van der Waals epitaxy n with complete ordering, and the chains are stacked by van der Waals interactions to form hexagonal crystals [1/2Ag1/2CN nanocrystals, the orientation of 1D chains is parallel to a shorter diagonal of the nanocrystal rhombus. Aligned Au and Ag atoms make lattice lines perpendicular to the 1D chain direction, which are parallel to a longer diagonal of the rhombus.The above observations indicate that Au or AgCN ,27. Seco or AgCN ,20,21,29ositions  resultedcrystals . In the 1/2Ag1/2CN nanocrystals is that the nanocrystals have preferential growth directions that are related to the underlying graphene lattice structures. In the left area of 1/2Ag1/2CN nanocrystals (circled in yellow) show good orientational alignment to the second-order diffraction peaks—(1–210) peaks—of graphene (circled in red), which means that 1D chains of Au1/2Ag1/2CN are preferentially aligned to the zigzag lattice directions of the underlying graphene in real lattice space. Because the 1D chains of Au1/2Ag1/2CN are parallel to a shorter diagonal of the nanocrystal rhombus, longer diagonals of the nanocrystal rhombuses are aligned to the armchair lattice directions of graphene.One of the most interesting points in the Au1/2Ag1/2CN can grow preferentially on chemically inert surfaces of graphene, and the second is what makes the alignment between the two materials. The first question can be answered with van der Waals epitaxy [1/2Ag1/2CN chain are bound to each other with strong covalent bonds, whereas the chains are held together via van der Waals forces [1/2Ag1/2CN crystal can be easily cleaved along the chains without producing any dangling bonds on its surface. Because this surface can generate van der Waals epitaxial interaction with pristine graphene surfaces, Au1/2Ag1/2CN nanocrystals can grow preferentially on graphene. It is noteworthy that in van der Waals epitaxy [1/2Ag1/2CN and graphene  can be uncommon [The above observations raise two questions: the first is how Au epitaxy ,23, whics forces . Therefo epitaxy ,23, lattgraphene . The secand WTe2 ,19,20,21uncommon ,5,6,7, a1/2Ag1/2CN nanocrystals synthesized on graphene. 1/2Ag1/2CN nanocrystals, we capture scanning electron microscopy (SEM) images without and with sample tilting  is maintained in different samples synthesized under an identical condition. The second case . Although additional experiments are needed to reveal detailed relationships between synthesis conditions and nanocrystal geometries, One of the notable geometrical characteristics of the Auat 80 °C c, by dryt 150 °C b,e,h, anat 80 °C c,f,i. Frnditions g–i. The nditions a,d,g whonditions b if we cond case b,e,h usiond case c,f,i usi1/2Ag1/2CN during the precipitation process. In the original synthesis condition  is presented in 1/2Ag1/2CN to metal nanocrystals. The beginning of the movie shows a crystalline area that has a lattice spacing of 5.16 Å, which is a characteristic spacing of Au1/2Ag1/2CN. This lattice spacing is clearly larger than the typical lattice spacings of Au or Ag FCC (face cubic centered) crystals, which are ~2.3 Å, ~2.0 Å, and ~1.4 Å from (111), (200), and (220) planes. Therefore, Au1/2Ag1/2CN can be easily identified with the lattice spacing of 5.16 Å in high-resolution TEM observations. The Au1/2Ag1/2CN crystal in the first frame of the movie is decomposed during the TEM observation. The snapshot at 15 min contains no lattice spacing of 5.16Å, and Au1/2Ag1/2CN peak intensities over time in FFT images  by atomic attachment as well as nanoparticle coalescence. The metallic nanocrystals have the above-mentioned lattice spacings , and would be bimetallic FCC nanocrystals composed of Au and Ag. The lattice patterns of bimetallic nanocrystals are clearly shown in 1/2Ag1/2CN decomposition and bimetallic nanocrystal growth are observed with in situ TEM. The lattice patterns of bimetallic nanocrystals are randomly orientated, and do not show any epitaxial alignment along the underlying graphene unlike their precursor material (Au1/2Ag1/2CN). It has been shown that metal cyanide complexes can be easily decomposed in the solid phase because the ionic bonding between metal cations and CN− ions is relatively weak [2, the Au atoms hold together to form Au nanocrystals [1/2Ag1/2CN decomposition process discovered in this study can be described as follows: During the TEM imaging of the AuT images b of TEM ely weak . These fely weak ,26,32. Rely weak ,31,33. Iely weak ,29. Beca1/2Ag1/2CN nanocrystal (radiolysis) to zerovalent Au or Ag atoms and (CN)2 gas. Then, the densities of Au and Ag atoms increase locally near the imaged Au1/2Ag1/2CN nanocrystal, and the reduced metal atoms aggregate to form AuAg alloyed nanocrystals. We envision that this newly-discovered process can be a good model system to investigate atomistic mechanisms of the early-stage process of metallic alloy formation, as in situ TEM observations of the AuCN decomposition process to Au nanocrystals delivered mechanistic understandings of crystal nucleation at the atomic level [The electron beam for TEM imaging acts as a reducing agent to decompose the Auic level .1/2Ag1/2CN nanocrystals on pristine graphene surfaces. The nanocrystals synthesized through simple drop-casting are crystallographically aligned to lattice structures of the underlying graphene. This epitaxial alignment would originate from the potential energy landscape of graphene’s hexagonal lattices similar to other metal cyanides, and experimental investigations on 3D structures and synthesis conditions imply that the rhombic 2D geometries originate from different growth rates depending on orientations along and perpendicular to 1D Au1/2Ag1/2CN chains. In addition, in situ TEM observations show the decomposition process of Au1/2Ag1/2CN nanocrystals to Au and Ag alloy nanocrystals under electron beam irradiation. These results provide an additional example of 1D cyanide chain families that forms ordered nanocrystals epitaxially aligned on 2D materials and reveal basic physical characteristics of the rarely investigated nanomaterial, Au1/2Ag1/2CN.In this study, we have presented the epitaxial growth of diamond-shaped Au


Transcranial sonography (TCS) is a convenient tool for detecting certain brain diseases, such as brain tumors. Few studies have reported on the use of TCS in the area of Sella turcica. The accuracy and repeatability of Sella turcica with or without pituitary tumor is not clear.This study aimed to investigate the feasibility and accuracy of TCS to measure the size of Sella turcica according to the measurement in MRI and determine its diagnostic performance in individuals with pituitary tumor.t-test or Mann–Whitney test, using the receiver operating characteristic (ROC) curve to analyze the diagnostic value of D1 in TCS for pituitary tumor.In this cross-sectional comparative study, healthy volunteers and patients with pituitary tumor were enrolled for examination of TCS and MRI between October 2020 and July 2021. The transverse diameter  of Sella turcica and the volume of the pituitary tumor were measured by TCS and MRI, respectively, and compared by using Student’s P = 0.054). In patients with pituitary tumor, the mean D1 was 2.0 ± 0.65  by TCS and 2.42 ± 1.0  by MRI (P = 0.000). The median measurement volume was 4.41 and 6.59 cm3 in TCS and MR, respectively (P = 0.000). The mean D1 was 1.31 ± 0.35 in healthy volunteers and 2.0 ± 0.65 cm in patients with pituitary tumor (P = 0.000). In the ROC curve analysis, the area under the curve was 0.836, and the optimal cutoff value (1.56) exhibited a sensitivity and specificity of 67.31 and 88.0%, respectively.A total of 75 healthy volunteers and 51 patients with pituitary tumor were evaluated. In healthy volunteers, the mean D1 was 1.30 ± 0.35  by TCS and 1.32 ± 0.29  by MRI (The consistency between the two imaging technologies performed well in D1 measurement, while the volume of the pituitary tumor was smaller as assessed by TCS than by MRI. D1 in TCS had good diagnostic performance in pituitary tumor. The Sella turcica is a saddle-shaped structure in which the anterior and posterior walls are formed by a part of the sphenoid bone. The cavernous sinus forms its lateral wall, which is traversed by the internal carotid artery. We found that transcranial sonography (TCS) can detect the Sella turcica structure which appeared as a hyperechoic ring. However, little literature has reported these features.The pituitary gland is surrounded by the Sella turcica. In normal populations, the pituitary volume has been found to increase with age . After 2An enlarged pituitary gland is associated with pituitary tumors, traumatic brain injury, and disorder of functions . PituitaTranscranial sonography is a fast, portable, repeatable, and cost-effective examination with which it is possible to observe the morphological appearance of the Sella turcica and the pituitary tumor directly by the transtemporal bone window, which is not mentioned in other literature as far as we know. The purpose of this study was to investigate the accuracy of the measurement and diagnostic performance of TCS on Sella turcica and pituitary tumors.Patients with pituitary tumors and healthy volunteers were recruited in our study from Beijing Tiantan Hospital between October 2020 and July 2021. The age of all subjects was greater than 26 years old, as the size of the Sella turcica is stable after this age , 9. PatiThis study was approved by the Research Ethics Board of Beijing Tiantan Hospital, Capital Medical University, and the requirement for written informed consent was waived.3) = anteroposterior diameter × superior–inferior diameter × left–right diameter × 0.52, both in TCS and MRI.We define the transverse diameter  and the anterior–posterior diameter  of the Sella turcica as the inner side of two hyperechoic lines between the cavernous internal carotid artery and the bony anterior and posterior wall, respectively, in the axial position Figure 1After enrollment, the sex and the age of each patient were recorded. D1 and D2 in all patients were measured both in MRI (T2-weighted MR images) and TCS, respectively, by two experienced radiologists who were blinded to the clinical and MR information of the patients when performing the TCS examination. Three diameters were measured in patients with pituitary tumor, and then the volume was calculated according to the formula given above. The TCS characteristics of the pituitary tumors were described through echogenicity and the integrity of the Sella bone.All patients underwent transcranial ultrasound examination by a phased-array ultrasound system equipped with a 1.6–2.5 MHz transducer . A dynamic range of 45–55 dB and a penetration depth of 14–16 cm through the transtemporal bone window were used . Image bFirstly, with the patient lying in a supine position, the plane of the mesencephalic brainstem is insonated through the transtemporal bone window parallel to the imagined orbito-meatal line. By tilting the probe slightly downward for about 10-20°, the Sella turcica plane is reached. For assessing the pituitary tumor, the probe was tilted upward slowly to find the maximum plane of the tumor and measure the anteroposterior and left–right diameter, and then the probe was rotated 90°clockwise to measure the superior–inferior diameter.2; voxel size = 0.6 × 0.6 × 5.0 mm3; matrix = 384 × 300. All scans were acquired without an intravenous contrast agent and with the patient in a supine position during end inspiration.The T2-weighted images of all individuals were obtained with a Trio 3.0T scanner (Siemens). The T2-weighted image parameters were as follows: TR = 5,800 ms; TE = 110 ms; flip angle = 150°; field of view = 240 × 188 mmt-test or Mann–Whitney test. Paired quantitative data were compared using paired Student’s t-test or Wilcoxon test.We recorded the measured values of the two radiologists separately and then took the average of their measurements as the final statistical data. To evaluate intra- and interoperator reproducibility, the interclass correlation coefficient (ICC) was calculated using both TCS and MRI measurements of D1 and PV. Measurement reliability was classified according to common criteria as excellent (ICC > 0.75), good (ICC = 0.60–0.75), fair (ICC = 0.40–0.59), and poor (ICC ≤ 0.40) . QualitaP-values less than or equal to 0.05 were considered statistically significant for all two-sided tests.Calculated ® SPSS® Statistics Version 26) statistical software was used for all data analyses. Receiver operating characteristic (ROC) curve analysis for predicting pituitary tumor by D1 was performed using MedCalc software .SPSS  were excluded because of a poor transtemporal bone window Figure 2P = 0.053), respectively, which subsume five empty Sella and three Rathke cyst. The median distance of D1 was 1.36 in women and 1.20 in men (P = 0.051).There were 32 women (42.7%) and 43 men (57.3%) in this group. The mean age was 51 ± 12 years. The anterior–posterior hyperechoic lines cannot be visualized by TCS in most cases, both in patients with pituitary tumor and in healthy volunteers, so we only compared D1 between the two groups. The average distance of D1 was 1.30 ± 0.35  by TCS and 1.32 ± 0.29  by MRI (P = 0.000). The median measurement volume was 4.41 and 6.59 cm3 in TCS and MR, respectively (P = 0.000).The cohort consisted of 22 women (41.5%) and 28 men (58.5%), with a mean age of 53 ± 12 years. Five tumors were not visualized because of pituitary microadenoma, and the remaining 45 were detected (90%). The pituitary tumors Figure 3P = 0.000).The average distance of D1 was 1.31 ± 0.35 in healthy volunteers and 2.0 ± 0.65 in patients with pituitary tumor .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.


Orientia tsutsugamushi which is endemic to the Asia-Pacific region. Infected adults rarely present with sensorineural hearing loss and otalgia. Though few cases of pediatric cases are known to present with otalgia, no pediatric cases of sensorineural deafness complicating ST have been reported to date.Scrub Typhus (ST) is an acute, febrile zoonotic disease caused by the bacterium Orentia tsutsugamushi IgM antibodies on rapid diagnostic test and the presence of chigger mite in the right external auditory canal on repeat examination. She responded dramatically to the empirical treatment of ceftriaxone and doxycycline. On follow-up, she did not have any residual hearing loss and her ear pain had resolved completely.A 5-year-old, previously healthy girl presented with a one-week history of high-grade intermittent fever, Headache and right ear pain with a recent onset of reduced hearing. She had a fever up to 39 °C, cervical lymphadenopathy, bilateral pleural effusion with diffuse infiltrations, and mild hepatosplenomegaly with no evidence of rash. Her initial examination was normal except for mildly enlarged tonsils. Unilateral right ear hearing loss was noted using Weber’s test. Evidence of progressive, mild anemia, and raised inflammatory markers was noted. Diagnosis of scrub typhus was confirmed by positive detection of Acute onset hearing loss or severe otalgia with or without findings should be an important diagnostic clue for suspecting scrub typhus in patients who present with a history of fever especially in endemic areas. Orientia tsutsugamushi. The trombiculid mites (genus Leptotrombidium) act as the vector and the disease is transmitted to humans through the bite from the infected larval stages (chiggers) of the mites [Rickettsia rickettsii can present with sensorineural hearing loss and otalgia [Scrub typhus (ST) is a mite-borne rickettsial zoonosis caused by he mites . ST preshe mites . Neurolohe mites –5. In ra otalgia –8. No peWe report a case of ST in a child presenting with severe otalgia and unilateral sensorineural hearing loss, an uncommon presentation of this neglected tropical disease.A 5-year-old girl, a previously healthy child, presented with a one-week history of high-grade intermittent fever, headache, and right ear pain. She complained of paroxysmal right ear pain but without any history of ear discharge, tinnitus, dizziness, or seizures. She had a headache with nausea but no vomiting or loss of consciousness. This was associated with some abdominal distension and pain. Her bowel and urine output was normal, however, she had dysuria. The patient’s mother denied any history of insect or tick bite even though the child usually plays in the fields during the day. Both parents are farmers.On the 7th day after the onset of illness, she complained of reduced hearing in her right ear without tinnitus, which gradually progressed to unilateral hearing loss.Physical examination was notable for fever of 39 °C and cervical lymphadenopathy. There was no evidence of any rash or eschar. Examination of the throat revealed mildly enlarged tonsils without signs of inflammation. Initial otoscopic examination of the ears was unremarkable. Unilateral right ear sensorineural hearing loss was noted using Weber’s tuning fork test since audiometric tests weren’t available at our district hospital. She was mildly tachypneic but did not require oxygen. Bilateral pleural effusion was noted. Abdominal examination was notable for mild hepatosplenomegaly.3), mild anemia (hemoglobin 10.6 g/dL) and slightly raised neutrophils (70%). Subsequently, over the next few days, her platelet counts gradually decreased to a nadir of 47,000 cells/mm3. The C-reactive protein was 105 mg/dL with an increased erythrocyte sedimentation rate of 20 mm/hr. Urine revealed mild proteinuria. Chest X-ray showed bilateral pleural effusion with bilateral infiltrates  in our setting [Laboratory tests revealed thrombocytopenia  manifestations are an uncommon and atypical feature of ST. Published case reports on CNS manifestations of ST in the pediatric population are limited. Among these, meningitis and meningoencephalitis are commonly reported with a subset of severe forms of patients presenting with altered sensorium and seizures . In 2014Otalgia with sensorineural deafness is an unusual manifestation of ST. To date, no pediatric cases of sensorineural deafness complicating ST have been reported. Most of these cases described in the literature are in adult patients , 8, 16. Our patient presented with nonspecific fever, headache, and associated sensorineural hearing loss, and severe otalgia. A sensory neural type of hearing loss has been reported in patients with rickettsial infections , 8, 17. In previous case reports in adults, otalgia usually appeared within the first week, followed by hearing loss and tinnitus in the second week after the onset of ST , 8. CompTimely diagnosis and effective antibiotic treatment are effective in achieving good clinical outcomes . HoweverClinicians working in endemic areas must consider ST and other rickettsial diseases in patients presenting with fever and sensorineural hearing loss or severe otalgia with or without otoscopic abnormalities.