To provide clinical guidance in hearing aid prescription for older adults with presbycusis, we investigated differences in self-reported hearing abilities and hearing aid effectiveness for premium or basic hearing aid users. Secondly, as an explorative analysis, we investigated if differences in gain prescription verified with real-ear measurements explain differences in self-reported outcomes. The study was designed as a randomized controlled trial in which the patients were blinded towards the purpose of the study. In total, 190 first-time hearing aid users (>60 years of age) with symmetric bilateral presbycusis were fitted with either a premium or basic hearing aid. The randomization was stratified on age, sex, and word recognition score. Two outcome questionnaires were distributed: the International Outcome Inventory for Hearing Aids (IOI-HA) and the short form of the Speech, Spatial, and Qualities of Hearing Scale (SSQ-12). In addition, insertion gains were calculated from real-ear measurements at first-fit for all fitted hearing aids. Premium hearing aid users reported 0.7  scale points higher total SSQ-12 score per item and 0.8  scale points higher speech score per item, as well as 0.6  scale points higher qualities score compared to basic-feature hearing aid users. No significant differences in reported hearing aid effectiveness were found using the IOI-HA. Differences in the prescribed gain at 1 and 2 kHz were observed between premium and basic hearing aids within each company. Premium-feature devices yielded slightly better self-reported hearing abilities than basic-feature devices, but a statistically significant difference was only found in three out of seven outcome variables, and the effect was small. The generalizability of the study is limited to community-dwelling older adults with presbycusis. Thus, further research is needed for understanding the potential effects of hearing aid technology for other populations. Hearing care providers should continue to insist on research to support the choice of more costly premium technologies when prescribing hearing aids for older adults with presbycusis.Clinical Trial Registration:https://register.clinicaltrials.gov/, identifier NCT04539847. Hearing loss is one of the most common chronic health conditions today . AccordiPresbycusis is described as the cumulative effect of aging resulting from a degeneration of the cochlea and characterized by reduced hearing sensitivity and speech understanding in noisy environments . The audHearing aids (HA) are the conventional choice of rehabilitation for older adults with presbycusis , and theversus basic-level HA technologies. From the HA users’ point of view, it is important to know whether there is evidence suggesting greater benefit with premium compared to basic HA technology. In other words, in the decision process it is important to know, if a premium-level HA is worth the cost, which is a highly relevant topic from both a clinical and a commercial perspective.There is a lack of knowledge regarding what level of technology should be recommended for patients with a hearing loss, and further research is needed to clarify the relative benefits of premium-level Real-ear insertion gain can be used to verify the actual gain provided by the HA and is defined as the sound pressure level (SPL) near the eardrum when aided, minus the SPL at the eardrum when unaided . VariatiClinical assessments of HA performance are often not predictive of real-world outcomes which underlines the importance of assessing the perceived outcome of the patients. Real-world outcomes of a HA fitting can be assessed using different methods, and one reasonable way would be to ask for the HA user´s opinion. Thus, the patient’s perspective has been argued to be the gold standard to assess HA effectiveness . Over thversus basic HAs among 25 and 45 older individuals (mean age 70 years), respectively, with mild-to-moderate bilateral hearing loss and included both first-time and experienced HA users  project that aims to improve audiological rehabilitation in Denmark and worldwide through an evidence-based renewal of clinical practice. Data in the BEAR study were collected from the Department of Audiology at Odense University Hospital (OUH), Region of Southern Denmark and the Department of Audiology at Aalborg University Hospital (AAUH), North Denmark Region from January 2017 to May 2018. Adults (≥18 years of age) referred for public HA treatment were enrolled in the BEAR study, regardless of previous HA experience. The BEAR project was evaluated by The Regional Committees on Health Research Ethics for Southern Denmark (S-20162000-64), and the present study was registered .Data were collected from 1,159 patients with hearing loss who were a subgroup of patients enrolled from Region Southern Denmark accepting to participate in the BEAR project . These patients were distributed across the whole BEAR study period. They were recruited by private ear-, nose-, and throat (ENT) physicians that were informed about the study, and project information letters were sent to the ENTs to distribute to the patients. All patients received a letter including information about study details, a consent form, and a note on the patient´s rights related to study participation. The consent to participate was forwarded in a referral letter to the Department of Audiology, OUH. The inclusion criteria for the current study were patients with bilateral presbycusis with no previous experience with HAs. Presbycusis was defined as a symmetrical hearing loss (less than 10 dB difference in PTA-4 between right and left ear) in patients older than 60 years where the high-frequency hearing loss were greater than the low-frequency hearing loss, and no disclosed history of hearing loss besides age. Patients were excluded if they were not native Danish speakers, not able to complete the consent form or the questionnaires, or if they were experienced HA users. Each patient’s audiogram and general medical history was contained in the referral letter from the private ENT specialists and checked by the responsible researcher. If the inclusion criteria were met, patients were randomly assigned to either a premium-feature or basic-feature HA, stratified on age , sex, and word recognition scores (WRS) (WRS≥80% and WRS<80%). Block randomization with varying block sizes (12 and 18) was managed in the electronic REDCap database enabling equal distribution of the stratification variables in the two groups. Allocation was concealed.n = 115/116)  . Twenty-115/116) . PatientSix different pairs of commercially available HAs  were evaluated as examples of basic-feature and premium-feature technologies from three manufacturers contributing to the BEAR project. One pair of basic-feature and premium-feature HA was included from each manufacturer. The HAs were released in different years and used different platforms, and the fitting strategy in one of the three companies differed between the two levels of HA technology, which is reported in All patients were bilaterally HA fitted by an experienced audiologist according to standard clinical practice, and the HAs were linked to the fitting software with wireless communication. The fitting and fine tuning of HAs were carried out in a single session following hearing evaluation. HAs were fitted using the proprietary fitting rationales by the specific HA manufacturers with NAL-NL2 target gains only being used for reference purposes in the analysis. Based on feedback from patients, If necessary, some gain adjustments in the high-frequencies were carried out to achieve a fit acceptable to the HA user. The decision about the acoustic coupling was based on individual characteristics of the ear canal and recommendations in the fitting software. All feature settings were set according to the recommendations of the manufacturer and therefore left in default setting. As all patients were first-time HA users, no additional programs were added to the default listening program, and patients were given a short instruction on how to use the HAs, clean the earmolds, and changing of batteries. If connectivity was available for the given HA model, the HAs were connected via Bluetooth. Patients in need of assistive listening devices (e.g. remote controls) were referred to the responsible agencies or personnel. They were all recommended to use the devices during waking hours for as long as possible. Besides, they were informed of the opportunity to ask for additional counselling if needed.idainstitute.com): ‘How important is it for you to improve your hearing?’ and ‘How much do you believe in your ability to use hearing aids?’. The scale is ordinal and ranges from 0 to 10. A higher score indicates higher motivation .All patients completed a questionnaire survey 2 weeks before the first visit to the clinic and 2 months after HA fitting. The outcomes were designed to capture the perceived hearing abilities and the effectiveness of HAs and included the SSQ-12  and the The questionnaire survey was managed by the research electronic data capture (REDCap) software that was developed by Vanderbilt University, Nashville, Tennessee, United States and hosted by Odense patient explorative network (OPEN) in the Region of Southern Denmark ; 2019. AAs part of the first visit to the clinic, all patients underwent standard audiometry according to current clinical practice. The audiological assessment included a pure-tone audiometry measuring air-conduction thresholds for left and right ears at 250 Hz, 500 Hz, 1 kHz, 2 kHz (3 kHz), 4 kHz (6 kHz) and 8 kHz; bone-conduction thresholds at 250 Hz to 4 kHz when air-conduction thresholds showed low-frequency hearing thresholds >20 dB hearing lossor were asymmetric between the two ears; and a measure of WRS. Tympanometry was measured to rule out any middle-ear diseases. Air- and bone-conduction thresholds were measured according to ISO8253-1:2010 . TDH39 headphones, or ER-3A insert earphones, were used during the tests. The WRS was obtained by presenting 25 different monosyllabic words in quiet at the most comfortable listening level from the validated DANTALE-I wordlists . The result is expressed as the percentage of correct responses to the words presented. All measurements were conducted in a soundproof booth in the Audiological Department at OUH and were carried out by experienced audiologists.A follow-up appointment was scheduled approximately 2 months after HA fitting. Real-ear measurements were performed both before and after any adjustments of the HAs and included measuring real-ear unaided gain at 65 dB SPL to record the natural gain provided by the outer ear followed by measuring real-ear aided gain at three different input levels . Finally, the real-ear insertion gain was derived by subtracting the unaided gain from the aided gain. Only the REMs that were obtained before any adjustments of the HAs were used in the analysis. The International Speech Test Signal (ISTS) was used as the stimulus, and the HAs were set to default so that all features were activated during the measurement. The REM module (REM440) of Affinity 2.0 (Interacoustics) was used and followed the standards: ANSI/ASA S3.46 (2013); IEC 61669 (2015); ISO 12124 (2001). Calibration of the REM headset was repeated before each session. The REMs were only used for documentation and not as a basis for adjusting the HAs. Hence, the NAL-NL2 fitting prescription was only used as a hypothetical reference target. In addition, the HA usage time in hours per day was extracted from the fitting software. Some of the logged data showed usage time of >18 h per day. This could have been due to patients not turning off the HAs during the night, and these data were therefore excluded. The follow-up visit was carried out by the two researchers at the Department of Audiology at OUH responsible for collecting the outcome data.p < 0.05 for the MLR analyses (command regress in STATA). Variance inflation factor (VIF) was used to test multi-collinearity between independent variables in all linear regression models and showed no indications for multi-collinearity in the models (VIF<2.5). The dependent variables used in the models were: IOI-HA total score, IOI-HA factor 1 and factor 2 score, SSQ-12 speech, SSQ-12 space, SSQ-12 sound quality score, and SSQ total score. The primary predictor used in the analyses was HA technology level, and secondary predictors were sex, age, WRS, motivation (Q1 and Q2), baseline SSQ-12 scores for the given domain, and mean gain deviation from NAL-NL2 targets at 1, 2, and 4 kHz. As an extended model, it was investigated whether the difference in manufacturer was a significant factor related to the outcome. The significant results from parametric analysis were checked and confirmed with non-parametric analysis using Mann-Whitney U tests as well.PTA-4 was calculated as the average of hearing thresholds at 0.5, 1, 2, and 4 kHz for each ear. Data normality was verified using Q-Q plots. Multiple linear regression (MLR) analyses were used to test the hypothesis that premium-feature HAs yielded better self-reported outcomes than basic-feature technologies in terms of overall SSQ score, SSQ domain scores, IOI-HA total score, and IOI-HA factor 1 and factor 2 scores. Baseline SSQ-12 scores were included in the models with SSQ-12 as an outcome to adjust for the unaided score, thereby minimizing the effect of individual differences that existed before HA treatment. Using linear regression as statistical model, the outcomes are treated as an interval scale despite ordinal scale properties. Since ordered logistic regression did not show any significant differences to the model estimates, linear regression was used in the analyses. Bootstrapping was applied to compensate for non-normally distributed residuals. Significance levels were set at Data management and analyses were performed using STATA SE version 16.0 .n = 190 and power = 0.80, we can detect a difference in IOI-HA total score of 1.91. Alpha was set to 0.05.Given an observed standard deviation (SD) of 1.54 and 1.61 scale points for the overall SSQ-12 score in the two study arms, a sample size of n = 190 and a power = 0.80, we can detect a difference in the overall SSQ-12 of 0.64. Also, based on the SD of 4.39 and 4.94 scale points for IOI-HA total score in the two groups and with the same sample size of n = 97/93). The median age of the two groups of HA users was 72 (interquartile range (IQR) 10) years and 70 (10) years, respectively. Forty-one to 45% were female HA users. The median WRS for the left and right ears of the two groups of HA users were 92% and the IQRs were 8% and 12%, respectively. The average PTA-4 levels for the left and right ears were 37.5 (12.5) dB HL and 36.4 (11.0) dB HL, respectively. The two groups were highly similar in terms of sex (p = 0.8), age (p = 0.2), PTA-4 right and left ear , WRS right and left ear , level of motivation Q1 and Q2 , and occupational status (p = 0.5). Also, there was no statistically significant difference in how much they used their HAs in hours per day in the two groups (p = 0.9).p = 0.96), factor 2 score , or IOI-HA total score  compared to basic-feature HA users. Using the SSQ-12 speech, space, and qualities domain score as outcome variables showed that premium-feature HA users reported a 0.8  scale points higher speech score per item in the speech domain and a 0.6  scale points higher qualities score per item in the qualities domain compared to basic-feature HA users. Using the SSQ-12 total score, results showed that premium-feature HA users reported a 0.7  scale points higher total score than basic-feature HA users. SSQ-12 differences similarly ranged from 0.5-1 between two different noise management settings in Andersson et al. (2021) whereas the differences between the two device technology levels in p = 0.02) scale points higher SSQ-12 score per item in the space domain, and those with higher motivation for HA treatment reported a 0.2  scale points higher SSQ-12 space score per one unit change in motivation score. WRS and motivation had similar significant effects on the total SSQ-12 score. Female HA users reported 0.7  units lower SSQ-12 space score per item in the space domain than male HA users. These results indicate that WRS, motivation, and sex can also modestly affect SSQ-12 scores.Patients with 10% higher WRS reported 0.4  between the premium and basic HA and for company B at 1 and 2 kHz  The mean difference between the insertion gains and NAL-NL2 target gains at the measured frequencies varied from 2 to 3 dB between the two levels of technology.One of the main findings from the current study was that presbycusis patients without previous HA experience using premium HAs reported better hearing abilities in terms of speech and qualities of hearing compared to those using basic HAs using the SSQ-12 as outcome measure. These results suggest that patients with symmetric presbycusis might benefit from premium technologies with regards to improved hearing abilities. Using the IOI-HA, showed no significant difference in reported HA effectiveness between the two levels of HA technology. In total, four out of seven outcome variables failed to show a significant difference between premium and basic HAs, and with larger samples sizes, even small effects can yield statistical significance. Hence, it is important to acknowledge the limited clinical relevance of the results. Also, given the rather small differences in SSQ-12 scores between the premium- and basic-feature HA users , it is questionable if these observed differences would be relevant to daily life. Although minimal clinically important differences have not been established for the SSQ-12, previous research has used one scale point of change to demonstrate a clinical significant change between assessments in SSQ ). HoweveIn contrast, previous studies did not provide evidence to suggest that premium HAs yield better self-reported real-world outcomes or laboratory outcomes . AlthougAnother important difference between previous studies and the current study is that the HA technology used in the previous studies was at least one developmental epoch older than the current study, and the technology advances could therefore be expected to be more evident in the premium devices used in the current study. Hence, it is noticeable, that even with greater contrast in technology, the IOI-HA total score along with the Factor 1 and Factor 2 scores failed to show a significant difference between premium and basic level HAs. This suggests that the perceived effectiveness using premium and basic technologies were similar. However, as the IOI-HA addresses the overall effectiveness with HAs and the SSQ addresses the hearing (dis)abilities in different specific listening situations, we speculate if the IOI-HA items were not specific enough to detect perceived differences between the two technology levels. Thus, the SSQ-12 appears to be a more sensitive outcome measure to uncover small differences in technology levels or feature settings.One explanation for premium HAs yielded better perceived hearing abilities in the SSQ speech domain could be that the more advanced features in the premium HAs can be better at distinguishing speech signals from noise than basic-feature HAs. The high-frequency boost feature and the speech-enhancer feature in premium HAs  could coFemale HA users reported significantly poorer hearing abilities than men in the space domain related to questions on directional and distance hearing. Previous studies have shown that females reported poorer outcomes than men using the IOI-HA which was related to Factor 2 scores (communication with others) , and theInsertion gain levels at first-fit measured with REM showed that premium HAs prescribed more high-frequency gain than basic HAs for company A, but the opposite was found for company B . The gaiAlthough most previous studies did not find real-world differences between premium and basic devices, individual characteristics such as noise acceptance and listening demands along with satisfaction in large groups showed to have an impact on the perceived benefit of different HA technologies . Those iOne of the aims of the BEAR project is to evaluate and improve the current hearing rehabilitation, including the HA prescription. Thus, to improve the clinical procedures in the HA provision process and achieve a personalized HA fitting, it is important that clinicians perform a thorough candidacy evaluation and consider the patients’ individual listening goals and auditory ecology, including the listening needs in their everyday life acoustic environments .This study aimed to investigate if arguments for prescribing a more costly premium HA for older adults with presbycusis could be found, thereby providing clinical guidance in HA prescription. We found only small differences on a subset of the self-reported outcomes between premium and basic level HAs. When HA cost is not covered, studies have shown that patients perform a cost-benefit analysis to decide if the premium HAs provide sufficient additional value to justify the extra cost. In this case, the willingness and ability to pay are important factors during the decision process. In countries were the HAs are free of charge as part of the public healthcare system, it is up to the clinician to decide if premium technologies are worth the extra cost, and therefore, the decision is based on the individual patient-clinician relationship. A further analysis of the cost-effectiveness of the HA treatment would be interesting but was beyond the scope of the present study.The main strength of the current study lies in its study design, which allows for a highly homogenous study population including only patients with symmetric presbycusis. This minimized the variation in patient demographics excludes the risk of different types of hearing loss to affect the results and is a clear advantage compared to other similar studies. In contrast to previous studies, the current study only included first-time HA users to exclude bias towards a certain HA model that would have been a potential risk using experienced users. In addition, this study investigated a larger study population, including 190 patients compared to 24, 25, or 45 subjects, respectively, included in previous studies . HoweverAn important limitation to consider is that the outcome measures used in this study reflect the patients’ perspective and might not be sensitive to the actual differences observed in their daily lives. Using self-reported measures such as the SSQ-12, we do not know if the addressed listening conditions were relevant or important to each HA user, or whether other relevant or important conditions were not addressed. A sentence-based test like HINT  or otherThe current study aimed to provide clinical guidance in HA prescription, to improve current clinical procedures for older adults with presbycusis. Premium HAs yielded slightly better self-reported hearing abilities in older adults with symmetrical presbycusis without previous HA experience, but this does not necessarily apply to other types of hearing loss, and the clinical relevance of the results is limited. Hence, there is limited evidence to support the choice of more costly premium technologies, and clinicians should therefore be careful not simply to conclude that more advanced technologies always produce a better outcome. Differences in real-ear insertion gain at first-fit did not explain the differences in reported outcome. Hearing care providers should continue to insist on evidence to support the choice of more costly premium technologies and include aspects as candidacy and auditory ecology in the HA provision to achieve a more personalized fitting and improve rehabilitation outcome.


Decreased handgrip strength (HGS) and activities of daily living (ADL) disability are common in aging populations. No studies have evaluated the bidirectional associations between HGS and ADL disability. This study aimed to explore the bidirectional effects of HGS and ADL disability.This study analyzed data from two waves (2011 and 2015) of China Health and Retirement Longitudinal Study (CHARLS). Low HGS is defined by the Asian Working Group for Sarcopenia criteria. Meanwhile, disability was assessed by ADLs scale. The prospective bidirectional association between HGS and ADL disability was examined using binary logistic regression. Subgroup analysis were performed according to age and gender.A total of 4,902 and 5,243 participants were included in the Stage I and Stage II analyses, respectively. On the one hand, low HGS was significantly associated with subsequent ADL disability. The odds ratio (OR) value of developing BADL disability and IADL disability were 1.60 : 1.23–2.08) and 1.40 (95% CI: 1.15–1.70), respectively, in participants with low HGS. On the other hand, baseline ADL disability was associated with an increased risk of developing low HGS. The OR value of developing low HGS were 1.84 (95% CI: 1.34–2.51) and 1.46 (95% CI: 1.19–1.79) for participants with BADL disability and participants with IADL disability, respectively. Lastly, the strength of the bidirectional associations varied among subgroups.A significant bidirectional associations were identified between HGS and ADL disability. Interventions should be developed to prevent the development or progression of both low HGS and ADL disability. Activities of daily living (ADLs) represent the fundamental skills required to care for oneself independently . ADL disMuscle weakness is another important event closely related to aging. Handgrip strength (HGS) has already been demonstrated as a reliable indicator of muscle strength . AccordiPrevious studies have investigated the association between HGS and ADL disability. For example, a cross-sectional study in India found that older adults with low HGS had a higher risk of experiencing functional difficulties with ADLs than older adults with strong HGS . SimilarIn this study, we used data from the China Health and Retirement Longitudinal Study (CHARLS) to (1) investigate the longitudinal association between baseline low HGS and subsequent ADL disability, (2) investigate the longitudinal association between baseline ADL disability and subsequent low HGS; and (3) examine the strength of these associations in different age and gender subgroups.CHARLS is a nationally representative longitudinal survey of middle-aged and older adults in China . The basThis current study analyzed CHARLS data obtained from the baseline (2011) and third wave (2015) data collection periods. A total of 9,833 participants were excluded due to (1) age < 45 years, (2) lack of information on baseline HGS and ADLs, (3) lack of information on demographics. In Stage I, after further exclusion of participants with baseline ADL disability and those lost to follow-up, 4,902 participants were included in the analysis. In Stage II, after further exclusion of participants with baseline low HGS and those lost to follow-up, 5,243 participants were included in the analysis. A detailed flowchart is presented in HGS was measured using an isometric dynamometer  . Staff dADL disability was measured using the basic activities of daily living (BADL) and instrumental activities of daily living (IADL) scales . BADLs r2, normal: 18.5–23.9 kg/m2, overweight and obese: ≥24 kg/m2) , address , and marital status (married or other). Lifestyle characteristics: smoking, drinking, sleeping time , napping time . In terms of health status, body-mass-index (BMI) was divided into three categories according to Chinese criteria (underweight: ≤ 18.5 kg/m4 kg/m2) . Partici4 kg/m2) .T-tests and Chi-square tests were used to compare baseline characteristics between groups. In Stage I, participants who were able to perform ADLs independently were divided into two groups based on baseline HGS  and evaluated in 2015 for independence in ADLs. In Stage II, participants with normal handgrip strength were divided into two groups based on presence/absence of ADL disability and evaluated in 2015 for handgrip strength. Binary logistic regression models were constructed to assess the prospective bidirectional association between low HGS and ADL disability. Odds ratios (ORs) and their 95% confidence intervals (CIs) were used to measure the strength of the associations. Multivariate models adjusted for age, sex, education level, address, marital status, smoking, alcohol, BMI, number of chronic diseases, history of falls, history of fracture, night sleep duration, nap duration, and depressive symptoms. In addition, subgroup analyses were performed based on age  and gender. Post-hoc power analyses were performed to guide interpretation of our findings  and counts (percentages), respectively. findings . All anaP < 0.001). Low HGS was also more common in underweight, low education, and other marital status  groups (P < 0.05). Other characteristics associated with low HGS included less sleep, a history of falls, and depression (P < 0.05). During the 4-year follow-up period, 104 (14.6%) and 205 (28.8%) participants from the low HGS group developed BADL disability and IADL disability, respectively. As shown in A total of 4,902 participants were included in the no baseline ADL disability cohort. These participants' baseline characteristics are shown in P < 0.05). In terms of health status, individuals with ADL disability had a higher number of chronic diseases, a history of falls or fractures, and depressive symptoms (P < 0.05). The detailed baseline characteristics of individuals with and without ADL disability are presented in At this stage, 5,243 individuals were included for analysis. Compared with individuals without ADL disability, those with functional disabilities were older and more likely to live in rural areas (Age and sex subgroup analyses were conducted for both analysis stages. As shown in This study determined that a bidirectional association exists between low HGS and ADL disability. Specifically, low HGS was found to predict the development of ADL disability, and ADL disability increased the risk of developing low HGS. These associations remained significant even after adjusting for confounding factors. Besides, the association between HGS and ADL disability varied by age and sex.Previous studies have focused on the effect of HGS on ADL disability. One Australian cross-sectional study found that for every 10 kg increase in HGS, the odds of ADL impairment decreased by 39% . SimilarTo the best of our knowledge, no study has investigated the effect of ADL disability on HGS. However, previous studies have investigated the effect of physical activity on HGS, wherein a lack of physical activity was found to lead to a decrease in HGS , 28. In The mechanisms that underlie the bidirectional association between HGS and ADL disability are likely complex and diverse. On the one hand, individuals with low HGS usually have higher levels of inflammation . Thus, iIn terms of subgroups, we found a stronger association between low HGS and subsequent ADL disability in women, which is supported by previous findings , 37. It This is the first prospective study to investigate the bidirectional association between low HGS and ADL disability. The study's strengths include a large nationally representative sample and longitudinal study design. However, some limitations present in the study should also be considered. First, some of the data were obtained through self-report, introducing the potential for recall bias. Second, the study design of CHARLS prevented us from obtaining multiple assessments of ADL disability status, which may be interfered with intermittent disability. Third, due to the limited number of individuals with ADL disability, the influence of specific ADLs on HGS is unknown and warrants exploration through future high-quality studies. Finally, although the overall sample was relatively large, a large number of participants were excluded due to a loss to follow-up and missing data, which may have resulted in selection bias.In conclusion, this study suggested a bidirectional association between HGS and ADL disability. Low HGS can be used as a reliable marker of future ADL disability, which in turn exacerbates the decline in HGS. It is necessary to strengthen the screening and intervention of low HGS and enhance the functional recovery of individuals with ADL disability to promote healthy aging. Moreover, age and gender should be considered when assessing the association between HGS and ADL disability to develop more accurate and effective interventions.The original contributions presented in the study are included in the article/The studies involving humans were approved by Biomedical Ethics Review Committee of Peking University. The studies were conducted in accordance with the local legislation and institutional requirements. The participants provided their written informed consent to participate in this study. No potentially identifiable images or data are presented in this study.SD: conceptualization and writing. SW: methodology. SJ: validation. DW: software. CD: investigation. All authors read and approved the final manuscript.


Generation expansion planning (GEP) is a complex, highly constrained, non-linear, discrete and dynamic optimization task aimed at determining the optimum generation technology mix of the best expansion alternative for long-term planning horizon. This paper presents a new framework to study the GEP in a multi-stage horizon with reliability constrained. GEP problem is presented to minimize the capital investment costs, salvage value cost, operation and maintenance, and outage cost under several constraints over planning horizon. Added to that, the spinning reserve, fuel mix ratio and reliability in terms of Loss of Load Probability are maintained. Moreover, to decrease the GEP problem search space and reduce the computational time, some modifications are proposed such as the Virtual mapping procedure, penalty factor approach, and the modified of intelligent initial population generation. For solving the proposed reliability constrained GEP problem, a novel honey badger algorithm (HBA) is developed. It is a meta-heuristic search algorithm inspired from the intelligent foraging behavior of honey badger to reach its prey. In HBA, the dynamic search behavior of honey badger with digging and honey finding approaches is formulated into exploration and exploitation phases. Added to that, several modern meta-heuristic optimization algorithms are employed which are crow search algorithm, aquila optimizer, bald eagle search and particle swarm optimization. These algorithms are applied, in a comparative manner, for three test case studies for 6-year, 12-year, and 24-year of short- and long-term planning horizon having five types of candidate units. The obtained results by all these proposed algorithms are compared and validated the effectiveness and superiority of the HBA over the other applied algorithms. Generation expansion planning (GEP) is an important decision-making activity in power systems which aims at deciding on new as well as upgrading existing generation units with different types. The decision should determine the features of where to allocate, when to install the new generation units, and what to select the new generation technology over a long-range planning horizon. The main objective function of GEP is to minimize the total costs which include the investment, operation and maintenance costs, and outage costs. At the same time, the upper construction limit, reliability, fuel mix, reserve margin constraints must be satisfied. GEP is a highly complex optimization problem due to including different nonlinear constraints. Therefore, the solution for this problem should be executed by more efficient methods to reach optimum solution3. GEP models are divided into two classes of static and dynamic. The static GEP model is a single period GEP problem which introduces less computational complexities of the problem, whereas dynamic GEP is a multi-period planning problem with higher computational complexity. Thus, dynamic GEP is more complex than static model.The power system is composed of three main sectors which are generation sector, transmission sector, and distribution sector. The planning issue of each element is a process that the investment decisions are made based on different aims to meet the forecast demand6. The GEP optimization frameworks with incorporation of renewable energy divides the various approaches into four categories: (a) conventional approaches that incorporate environmental limitations throughout the GEP; (b) formulating the GEP as a multiple-objective optimization problem; (c) strategies for addressing variable RES-related uncertainties in the GEP process; and (d) different dynamics and issues brought about in the power networks due to the growing incorporation of intermittent RES. It contributes to an improved comprehension of the anticipated results of each technique by offering insights into the traits, benefits, and drawbacks of the theoretical methods used as well as their applicability for various parts of the problem. Because transmission network operators as well as other managers believe that such models produce reliable results, the list of national/regional optimization models is lengthy.A few contemporary review publications are available that categorize the GEP models8. There are several tools to calculate the expected energy produced based on EENS. Therefore, the proper valuation of EENS is required to provide accurate estimates of the variable costs. Power system probabilistic production simulation (PPS) provides an accurate evaluation framework for EENS, and LOLP9. Also, several methods have been handled to solve such complicated optimization problem. Some of the classic optimization methods, that have applied to solve the GEP problem, include Analytic Hierarchy Process10, dynamic and quadratic programming12 and mixed integer nonlinear programming (MINLP) model14. In14, the option of Demand-Side Management (DSM) has been modeled as an equivalent generating unit which used only at peak load to maintain system reliability. In15, the GEP problem incorporating renewable energy has been presented where an hourly unit commitment problem has been used for supporting the selection of fuel mixes of power plants. In17, the least cost GEP model considering emission reduction has been presented that the penalty cost of emission was added to the objective function but the reliability constraint has not taken in account. Also, the GEP problem with the integration of renewable energy technologies and expected impacts in term of emission reduction has been presented in18. Despite the simple and easy implantation of MILP in solving the GEP problem, it has been utilized with low control variables. In19, MILP with fuzzy objectives has been applied to create the expansion model of the primary heat source in the district heating systems, combined heat and power units. In20, the GEP problem was handled by MILP considering the impact of unit commitment and renewable sources but the forced outage rate of generation units didn’t taken into account at expected energy produced calculation. However, because of GEP is a complex problem with nonlinear constraints, the convergence of those classical methods to the optimal decision makes difficult to solve. Also, there are commercial packages like Wien Automatic System Planning (WASP) Package has been used for solving GEP problem22. On the other side, the unavailability of generation units has not considered at variable costs calculations in21.In generating system analysis, the ability to meet the demand requirements is measured by the reliability criterion of the system which depends on the availability of generation units. The reliability of a system can be calculated by different indices such as loss of load probability (LOLP), loss of load expectation (LOLE), and expected energy not served (EENS). LOLE index is widely used, because of its simplicity. EENS reflects the true risk than LOLE so that it is an appealing index than LOLE. Also, the EENS index can be used to calculate the expected energy produced by each generation unit for each stage of the planning horizon23. This model was designed as a two-step process, with the first phase including dynamic programming to determine the best connection lines for electricity and the subsequent phase involving optimal transmission and GEP. A dynamic, integrated planning model for GEP involving transmission expansions was additionally created in24 using the linearized form of the AC power flow model as the basis for the generalized bender's decomposition approach. With the goals of minimizing risks at each level of expected return and maximizing anticipated return for any particular level of risk, Ref.25 adapted portfolio theory to the optimum GEP issue of Iran including financial risk management. Furthermore, with the aim of minimizing the projected cost and the conditional value-at-risk, Jin et al. presented a two-phase stochastic mixed-integer programming to handle the GEP issue under unpredictable growth in demand and fuel prices development26. Ref.27 explores the complementing possibilities of demand-side response by looking at researches on the German subsequent energy system that would exclusively use RES. In the example of the Chilean electrical grid, Ref.28 handles the GEP concerning substantial RES penetration by using a column generation technique and a unique Dantzig-Wolfe decomposition. The study shows that the suggested method beats commercial solver software because it surpasses intractability and drastically lowers computing load.In addition to that, a mixed integer linear programming model for the optimal GEP problem was introduced in Ref.31, gravitational search algorithm (GSA)32, shuffled frog leaping algorithm (SFLA)33, and modified shuffled frog leaping algorithm (MSFLA)9. The performance of GA was improved by application of Virtual mapping procedure, and controlled elitism (NSGA-II) which has been used for solving the GEP problem with and without network constraints considered as in31, respectively and compared with differential evaluation as in34. Also, investigation the impact of RES penetration on environmental aspect has been conducted over the GEP model using GA32. The implantation of capacity expansion planning based on electricity market was developed in30 to help the generation companies to take their decision whether to invest on new assets. In9, the comparison between GA, SFLA, and MSFLA has been presented to solve GEP problem with reliability constrained. Also, particle swarm optimization (PSO), differential evolution (DE), evolutionary programming (EP), ant colony optimization (ACO), tabu search (TS), simulated annealing (SA), and hybrid approach have been applied in35. In this study, the penalty factor of reliability criteria, which were used, was very small compared to the objective function which doesn’t guarantee the feasibility of the achieved solutions. So that, the output results haven’t been achieved the reliability constraint. Thus, the value of the penalty factor must be equal to several times of the objective function. PSO algorithm has been used in36 to solve the GEP problem in the deregulated electricity market. Also, a centralized GEP problem has been addressed by a distributional robust chance-constrained as in37.Over the last decades, there has been a growing concern in modern nature-inspired algorithms which are classified as meta-heuristic algorithms such as genetic algorithm (GA)33.The GEP dimensionality is large which the length of a string is equal to the product of the number of planning stages and the number of candidate unit’s types. So that the Virtual mapping procedure (VMP), penalty factor approach (PFA), and the modified of intelligent initial population generation (MIIPG) have been accomplished to decrease the GEP problem search space and reduce the computational timeA multi-stage GEP with reliability constrained is presented to minimize the capital investment costs, salvage value cost, operation and maintenance, and outage cost under several constraints.A comparative assessment for solving the proposed reliability constrained GEP problem is performed of different modern algorithms of PSO, CSA, AO, BES, and HBA.Several modifications are proposed such as VMP, PFA, and MIIPG to decrease the GEP problem search space and reduce the computational time.A high effectiveness and superiority of the proposed HBA over the other applied algorithms for solving the proposed reliability constrained GEP problem for three test case studies for 3-year, 12-year, and 24-year of short- and long-term planning horizon.The contributions of this paper with respect to the previous research in the area can be summarized as follows:The rest of this paper is organized as follows: in the next section, the GEP problem formulation is presented. In “Honey badger algorithm for reliability constrained dynamic GEP” section, the GEP problem modifications are described, the proposed HBA algorithm for solving the reliability GEP problem is described in “Test system description and results discussion” section. The test system and simulation results for the reliability GEP problem are presented in “Conclusions” section. Finally, the paper is concluded in section VI.33:Solving a reliability constrained GEP problem with minimum total costs aims at determining the optimum expansion plan over planning period that achieves minimum total cost under constraints satisfaction. The GEP total costs can be divided into two main parts which are related to new installed and already existed candidate units. In this context, the investment and salvage costs belong to the first part while operation and maintenance and outage costs are related to the candidate and existed units all together as followingThis term represents the investment cost of new candidate units k added in stage t which are selected of different technologies; t.where, 1.Salvation value This term of the objective function is the generation operation and maintenance costs for existing and new candidate units and assumed to occur in the middle of the corresponding planning stage which is calculated as follows:38. This method provides a relatively simple approach for EENS calculation but take more computation time, so that will not be suitable for GEP with the large-scale size problem. Therefore, another relative computational speed and solution quality of six different probabilistic production cost simulation methods have been presented for expected energy produced as in39. In this context, the comparison results have been implied that the probabilistic production cost simulation using the equivalent energy function (EEF) method is more accurate and takes less computation time.The expected energy produced by each generation unit in the system can be determined by the calculation of the expected energy not served (EENS). The EENS, which is one of reliability indies, can be calculated by conventional method or probabilistic production cost simulation methods to obtain cost modeling. The conventional method is composed of, establishing capacity outage probability table, load probability table and margin between them to calculate the reliability indies such as EENS, and LOLPThere are several reliability indices such as EENS, LOLP and LOLE. The EENS reflects the reliability status of a power system better than other indices. In this context, customer satisfaction with better supply will greatly influence the utility’s competitive ability. Hence, continuously energy supplying, which indicates better system reliability, can achieve customer satisfaction. On the other hand, each generating may be not available at any time depending on its forced outage rate (FOR). So that, the EENS cannot be made zero, but should be minimized as a cost term which can be formulated as follows:Upper construction limit: the number of each generation technology has committed that must satisfy the maximum construction number at stage t as follows:Spinning reserve constraintThe forecasted load demand and capacity reserve margin constraint must be met by the existing and new selected candidate units, which is represented as follows:Fuel mix ratioSelection of candidate units for expansion planning must be a limited ratio of each technology to all the existing units’ capacity that is expressed as following:Reliability constraint:The existing and new candidate units must satisfy the reliability criterion to maintain supplying energy continuously. LOLP is another reliability index to represent the system robustness in response to any contingencies.Several constraints must be considered during the expansion planning process which represented as follows:40. Also, EEF has been applied for estimating the EENS, LOLP, and expected energy produced for solving GEP problem as35 which is dependent on the system load duration curve (LDC) within a period of T as it is shown in Fig. As mentioned before, the calculation of expected energy produced by each generation unit, and reliability indices calculation are very important issues for solving GEP problem. For that purpose, probabilistic production simulation (PPS) methods have been widely used for such calculations. The PPS model based on EEF method has been established to analyze the impacts and benefits of efficiency power plant as inThe The FOR of the generation units is considered for the energy function that is represented for random outage of the generation units in EEF method. Suppose the generation unit i has a capacity of The above equation is the convolution formula in the EEF method, in which All the committed generation units have been sorted in ascending order based on their variable costs. Then, the energy function Because the GEP problem is a high dimensional problem with multiple conflicting objectives and nonlinear constraints which makes complex optimization problem, some modifications have been applied to deal with this problem. These modifications such as Virtual mapping procedure (VMP), penalty factor approach (PFA), and modified of intelligent initial population generation (MIIPG) have been used for facility the GEP problem and improve the effectiveness of the meta-heuristic algorithms as follows:33. The steps involved in VMP can be stated as follows:Form all the possible combinations of the candidate units.For each combination, multiply the number of units with the corresponding capacities and add them to get the total capacity of this combination.Arrange the total capacity in ascending order based on operation and maintenance costs.This mapping procedure transforms each combination of candidate units into a dummy variable for each stage. This dummy variable represents a position of each agent in the search space which is updated at each iteration based on using an algorithm. Thus, the decision variable of each stage is represented by single variable only which needs less memory space. Further, if the mapped variable took part in all related solutions, a small change in the mapped variable will reduce the infeasible solutionsAs a result, a multivariable could be reduced to a single variable, which in this case serves as the decision variable. The array size of the solution to the problem will grow by multiples of 5 because it is assumed that five distinct types of units will be used as choice variables at each stage. However, the decision variables obtained by using VMP are a multiple of the number of stages as the array size becomes 1 instead of 5 for each stage. Thus, a size reduction of 80% for each stage is realized. This reduces the dimensionality and the used memory space while improving the algorithm’s performance.33 are the case since their acquired reliability constraint is violated their permissible ranges.Because the constraints for GEP problem are nonlinear and more complex, obtaining feasible solutions is more difficult. However, the constrained problem can be transformed into an unconstrained problem by using the PFA, which is common for all the meta-heuristic techniques. Thus, the infeasible solutions are avoided in the subsequent iterations by adding proportional penalty values of each violated constraint to the objective function. The objective function with the PFA is given as the fitness function cost (FC):d in Eq. ;\document based on forecasted load demand and reserve margin. The preceding stage's minimum and maximum capacities, 2.The first step in any optimization problem is the creation of several populations randomly. Then, the position of each agent is updated according to the performed algorithm. Thus, many of initial solutions may be infeasible due to large search space and complex constraints that affected the convergence and performance of the algorithms. Therefore, the creation of initial population is incorporated to decrease the search space. The minimum and maximum cumulative capacity is considered in this procedure as having been made available in the earlier stages. As a result, the following formula can be used to determine the minimum and maximum capacity needed for each stage depending on reserve margin:41. It prefers to stay alone in self-dug holes and meet the other badgers only to mate. Because of their courageous nature, it can be attacked by even much larger predators when it cannot escape. Also, it can climb on trees for reaching bird nests and beehives for food. A honey badger locates its prey by smelling mouse skills and digging or follows the honey guide bird, which can locate the hives but cannot get honey. The first method of honey badger to reach its food source is called digging mode while the second method is called honey mode. The first mode is executed by honey badger alone, but the second strategy is executed with the help of other birds to locate the hives. The second mode phenomena lead to a relationship between the two which are enjoying the reward of teamwork. However, HBA has dynamic search modes, because of its ability to maintain the trade-off balance between exploration and exploitation in the searching process. The mathematical model of HBA is represented as follows:One approach of obtaining the optimal solution of the GEP problem is through the modern meta-heuristic optimization algorithms. Most of these algorithms are based on natural phenomena, where a fitness function is used as an indicator for the distance from the optimal solutions. The advantages of reducing array size by using VMP, converting the constrained problem into an unconstrained problem by using PFA. Also, the solution space reduction can be utilized by using MIIPG and implanted for solving GEP problem. For solving the proposed reliability constrained GEP problem, a novel honey badger algorithm (HBA) is developed. It is a meta-heuristic search algorithm inspired from the intelligent foraging behavior of honey badger to reach its prey. In HBA, the dynamic search behavior of honey badger with digging and honey finding approaches is formulated into exploration and exploitation phasesN) and their respective positions as follows:The first step of the proposed HBA is to initialize the number of honey badger based on the population size number  is generated which alters the search direction. Thus, agents now have several options for thoroughly exploring the search space. The equation that follows determines it:To enhance escaping from local to optima regions, a flag  is defined which is related to concentration, strength of the prey and distance between it and In the digging phase, a honey badger performs actions like Cardioid shape which can be simulated as follows:A honey badger follows the honey guide bird to the replicated beehive during the honey phase.ing Eqs.  and 26)29\docume41.Therefore, a honey badger searches near its prey, and the search is influenced by time variations  are are50. TCase-1: short-term GEP problems with 6-year planning horizonCase-2: long-term GEP problems with 12-year planning horizonCase-3: long-term GEP problems with 24-year planning horizonIn this section, the proposed HBA is performed for solving three cases based on the considered planning horizon as:Each planning horizon consists of two years of stage, making them to be 3, 6, and 12 stages in short and long-term planning horizon. Number of years between the reference date of cost calculations and the first year of study , coal and Nuclear (Nuc. PWR)) as in Table 15, is used to penalize the infeasible solutions.The utilized optimization algorithms of PSO, CSA, AO, BES besides the proposed HBA is handled for solving the short- and long-term capacity expansion planning based on 200 of iteration number, and 60 of population size for 30 simulation runs. In BES algorithm, each population of solution has been updated three times at each iteration where it is updated once for the other applied algorithms. Thus, the population size is 30 for BES only. Moreover, the penalty factor value  × 107, (2.99/16.4) × 107, (2.53/13.8) × 107, and (3.09 /16.9) × 107 respectively. These findings demonstrate the effectiveness of the HBA for solving the GEP in case-1 compared to the other algorithms.In case-1, the CSA, AO, BES, PSO, and the proposed HBA are employed for solving reliability constraint GEP over 6-years (3 stages) planning horizon. Table 9 $ where the CSA, AO, BES and PSO achieve 7.3464 × 109, 7.3267 × 109, 7.1976 × 109 and 7.2265 × 109 $, respectively. Also, the reliability criterion of LOLP obtained are shown in Table Moreover, Figs. 10$.The proposed HBA algorithm provides the smallest average objective with 1.3675 × 1010$.The proposed HBA algorithm provides the smallest worst objective with 1.4211 × 107$, compared to (6.32/3.46) × 107, (6.42/3.51) × 107, (6.87/3.76) × 107, (5.57/3.05) × 107 $ for the CSA, AO, BES, PSO algorithms.The proposed HBA algorithm provides the smallest standard error and standard deviation with (4.43/24.3) × 10In this case, a long-term planning horizon 12-year 6 stages) is conducted. CSA, AO, BES, PSO, and the proposed HBA are employed for solving reliability constraint GEP for this case. Table  stages iIn this context, the values of LOLP criterion of each stage, which must be satisfied, are also shown in Table 10 $.The proposed HBA algorithm provides the smallest average objective with 2.4953 × 1010 $.The proposed HBA algorithm provides the smallest worst objective with 2.6569 × 108 $, respectively which are smallest than that obtained by CSA, AO, BES, and PSO algorithms as (1.34/7.35), (1.48/8.13), (1.86/10.2), (1.34/7.35) × 108 $, respectively.The proposed HBA algorithm provides standard error and standard deviation with (1.33/73.1) × 10In this case, a long-term planning horizon 24-year (12 stages) is conducted. CSA, AO, BES, PSO, and the proposed HBA are employed for solving reliability constraint GEP for this case. Table In addition to, the reliability criterion is satisfied for each stage which the values of LOLP are given in Table 34 but the proposed study achieved greater reduction the investment costs. Moreover, the presented study based on the HBA addressed three different planning horizons of 6, 12 and 24 years. Otherwise the loss of load probability has been greatly minimized to 0.01 using the presented study based on the HBA and the elitist Non-dominated Sorting Genetic Algorithm version II29.To provide a comparison with literature work, Table This paper presents a novel honey badger algorithm (HBA) for solving the proposed reliability constrained Generation expansion planning (GEP) problem. In the GEP problem, a multi-stage model with reliability constrained is presented to minimize the total costs over the planning horizon maintaining several practical constraints of the spinning reserve, the fuel mix ratio and Loss of Load Probability. Also, the Virtual mapping procedure, penalty factor approach, and the modified of intelligent initial population generation are incorporated to the HBA to decrease the search space and reduce the computational time. Besides the proposed HBA, four modern meta-heuristic optimization algorithms were applied in three test cases to solve the short- and long-term reliability constraint GEP problem. They are crow search algorithm, aquila optimizer, bald eagle search and particle swarm optimization. The proposed HBA was successfully applied to reliability constrained GEP problems, with results that outperform CSA, AO, BES, and PSO in term of best, average, worst, standard error, and standard deviation. The proposed HBA achieves an order of magnitude of improvement for short- and long- term expansion planning than other algorithms. Future studies will be made to incorporate uncertainties such as load demand, and outage of generation units within GEP problem.Supplementary Tables.


At last, according to biotransformation rules of the prototype drugs in vivo, the secondary metabolites were traced and preliminarily chosen as Q-markers. As a result, 128 plant secondary metabolites were identified in HGT, and 11 specific plant secondary metabolites were screened out. Then, the content of specific plant secondary metabolites in 15 batches of HGT was determined, which confirmed their measurability. And the results of literature mining showed that eight secondary metabolites had therapeutic effects in treating liver disease at the in vivo level, and three secondary metabolites inhibited liver disease-related indicators at the in vitro level. After that, 26 compounds absorbed into the blood (11 specific plant metabolites and their 15 metabolites in vivo) were detected in rats. Moreover, 14 compounds, including prototype components and their metabolites, were selected as Q-marker candidates by the “TCM formula-botanical drugs-compounds-targets-pathways” network. Finally, 9 plant secondary metabolites were defined as comprehensive and representative Q-markers. Our study not only provides a scientific basis for the improvement and secondary development of the quality standard of HGT, but also proposes a reference method for discovering and identifying Q-markers of TCM preparations.Quality marker (Q-marker) serves as an important driver for the standardization of quality control in traditional Chinese medicine (TCM) formulas. However, it is still challenging to discover comprehensive and representative Q-markers. This study aimed to identify Q-markers of Hugan tablet (HGT), a famous TCM formula with ideal clinical effects in liver diseases. Here, we proposed a funnel-type stepwise filtering strategy that integrated secondary metabolites characterization, characteristic chromatogram, quantitative analysis, literature mining, biotransformation rules and network analysis. Firstly, the strategy of “secondary metabolites-botanical drugs-TCM formula” was applied to comprehensively identify the secondary metabolites of HGT. Then, the secondary metabolites with specificity and measurability in each botanical drug were identified by HPLC characteristic chromatogram, biosynthesis pathway and quantitative analysis. Based on literature mining, the effectiveness of botanical metabolites that met the above conditions was evaluated. Furthermore, the metabolism of the above metabolites  Corydalis Rhizoma as an example  are chemical components that associated with drug effects (such as effectiveness and safety), and are transferable and traceable components in the process of production and preparation . The con example . Zhang e example . Chen et example . In conc example .in vitro, which lack specificity and validity ; minister medicines, such as Artemisiae Scopariae Herba  and Pulvis Fellis Suis , that exert the efficacy of clearing away dampness and heat, relieving bile and reducing yellowness; assistant medicines, with liver-protecting, heat-clearing and detoxifying properties, containing Schisandrae chinensis Fructus  Baill.), Isatidis Radix  and Mung Bean  R. Wilczek) (Hugan tablet (HGT), a Chinese classic formula with definite clinical effect on liver diseases, is derived from Yinchenhao decoction and Xiaochaihu decoction, which has the effects of soothing liver, regulating qi, invigorating spleen as well as dissipating food . The preWilczek) . Our preWilczek) . NeverthWilczek) . StudiesWilczek) . Hence, Wilczek) . Currentin vitro to in vivo, a systematic study was conducted to excavate ingredients that could serve as Q-markers. Our study not only provided a new idea about identifying Q-markers in quality control research of TCM formulas, but also screened out reasonable Q-markers of HGT for the first time and improved its quality control level.Here, a funnel-type stepwise filtering strategy was proposed and applied to a research example of HGT : 1) The Reagents and materials are listed in the Two-month-old male SD (Sprague-Dawley) rats (200 ± 20 g) were purchased from Beijing Spelford Biotechnology Co., Ltd. , and were normally raised by the Experimental Animal Center of Beijing University of Chinese Medicine, in a specialized pathogen free (SPF) standard manner. After environment adaptation for 3 days, all rats were randomly grouped and fasted for 12 h before administration with drugs. The studies involving animals were reviewed and approved by the Animal Care and Ethics Committee of Beijing University of Chinese Medicine .n = 3): the clinical equivalent dose of HGT group, high dose of HGT group and representative compound groups -Goitrin, chlorogenic acid (CA), saikosaponin b2 (SS2), schisandrin (SSD), schizandrol B (SZDB), schisandrin A (SSDA), schisandrin B (SSDB) and schisandrin A (SSDC)). Before administration, all rats were fasted for 12 h and given water freely. On the one hand, the HGT solutions were intragastrically administered to the rats (n = 3) at 0.56 g/kg/d  and 7.00 g/kg/d (high dose) for 1 day. On the other hand, those representative compounds were orally administered to rats at a dosage of 20 mg/kg/d for 1 day, respectively. Medicated serum was collected from the orbital vein before and after oral administration at 5, 30, 60, 180, 360, 240, 480, 600 as well as 720 min, was centrifuged  and stored at −80°C.In the biotransformation profile characterization study, all rats were randomly divided into 10 groups . Then, the serum sample (1 ml) was treated with methanol (3 mL) and vortexed for 30 s to precipitate the protein. After centrifuging at 14,825 ×g for 15 min, the supernatant was evaporated to dryness under nitrogen gas at room temperature. At last, the residue was redissolved with 100 μl methanol, and the mixtures were vortexed for 30 s and centrifuged at 14,825 g for 10 min to obtain supernatant. The preparation method of the BR, ASH, SCF, IR, PFS, and MB samples was the same as the above.The HGT consisted of 0.2 g MB, 4.2 g BR, 4.2 g IR, 4.2 g ASH, 0.3 g PFS as well as 2.2 g SCF. The accurately weighed HGT powder  and 70% methanol (25 ml) were transferred into a 100 mL volumetric flask. Then, the mixtures were ultrasonically extracted for 1 h and centrifuged at 14,825 Twenty individual standard stock solutions were prepared by dissolving accurately weighed standards in methanol and stored at 4°C in the dark. Then, the mixed standard stock solution for characteristic chromatogram analysis was prepared by employing standard stock solutions of 20 compounds to reach the final concentration . In the -1 and the injection volume was 2 μl.The UPLC-Q-Exactive-Orbitrap/MS of Thermo Fisher Scientific corporation  was used for the identification of the chemical components in HGT and botanical drugs. The chromatography analysis was performed on a Waters ACQUITY UPLC HSS T3  at 40°C. The mobile phase consisted of 0.1% formic acid in water A) and acetonitrile B). The gradient elution with the following program was carried out: 0–10 min, 2% B–11% B; 10–15 min, 11 B%–20% B; 15–25 min, 20% B–30% B; 25–28 min, 30% B–40% B; 28–47 min, 40% B–98% B; 47–51 min, 98% B; 51–56 min, 98%–2% B; 56–60 min, 2%–2% B. The flow rate was set at 0.3 mL minAfter chromatographic separation, electrospray ionization (ESI) was selected to collect MS data in both positive and negative ion modes. The detailed parameter conditions were controlled as follows: the exact mass number was corrected with leucine enkephalin; ion spray voltage in positive ion mode, 3800 V; ion spray voltage in negative ion mode, 3200 V; capillary temperature, 350°C; sheath gas flow rate, 35 Arb; auxiliary gas flow rate, 15 Arb; m/z range, 100–1500 Da.18 column , and the column temperature was set at 30°C. The mobile phase was composed of 0.1% phosphoric acid aqueous solution A) and acetonitrile B) using a gradient program: 2%–20% B in 0–25 min; 20% B in 25–30 min; 20%–26% B in 30–35 min; 26%–40% B in 35–45 min; 40%–98% B in 45–75 min; 98% B in 75–80 min. The flow rate was controlled at 1 ml min-1, and the injection volume was 10 μl. The detection wavelengths were 200 and 250 nm.The characteristic chromatogram of HGT was constructed on a UPLC system  equipped with a PDA detector. Chromatographic separation was achieved by a Thermo Syncronis C-1) as well as different columns .The precision, repeatability and stability experiments were designed, evaluating the stability of the instruments and verifying the reliability of the data. Firstly, a HGT sample was prepared for six times consecutive analyses to assess the precision of the instrument. And the repeatability method was confirmed on account of the continuous analysis of six replicate HGT samples. Furthermore, analyzing one HGT sample at 0, 2, 4, 8, 12 and 24 h was to evaluate the stability of the method, respectively. Finally, to evaluate the method’s durability, chromatographic analysis of a HGT sample was conducted at different column temperatures , different flow rates , and the column temperature was controlled at 35°C. The 0.3% formic acid in water A) and acetonitrile B) were chosen as the mobile phase. An optimized elution condition was applied as follows: 0–10 min, 2%–7% B; 10–18 min, 7% B; 18–25 min, 7% B–15% B; 25–30 min; 15% B; 30–35 min, 15% B–20% B; 35–40 min, 20% B–26% B; 40–50 min, 26% B–40% B; 50–68 min, 40% B–69% B; 68–72 min, 69% B; 72–86 min, 69% B–98% B; 86–105 min, 98% B. The flow rate was 1 ml min-1, and the injection volume was 10 μl. The detection wavelengths were 200 and 250 nm.Chromatographic separation was performed on a Thermo Syncronis CThe validation parameters, involving specificity, linearity, precision, stability, reproducibility and recovery of 11 specific components in HGT, were systematically examined. On the basis of the average peak areas of 11 standard solutions at a range of concentrations, the linearities were constructed. And the calibration curves were plotted with the concentration of the homologous standard solution x) as a horizontal coordinate and the average peak area y) as a vertical coordinate. Then, a HGT sample was prepared for 6 times consecutive injections to validate the precision. Six replicate HGT samples in parallel were processed for repeatability evaluation. The sample solution was placed in the UPLC autosampler and analyzed at different periods  to assess the stability of the sample. The average recoveries of compounds were investigated by adding a number of standard solutions to 1.0 g HGT powder. Nine samples were prepared parallelly according to the part “Preparation of standard solution and sample solution”. Variations were indicated by percentage relative standard deviations (RSDs).-1. The gradient elution program was applied as follows: 2% B–11% B (0–10 min); 11% B–20% B (10–15 min); 20% B–30% B (15–25 min); 30% B–40% B (25–28 min); 40% B–98% B (28–47 min); 98% B (47–51 min); 98%–2% B (51–51.5 min); 2%–2% B (51.5–55 min). The injection volume was 2 μL.Chromatographic separation was conducted on a WATERS ACQUITY UPLC HSS T3  at 40°C. The mobile phase contained 0.1% formic acid aqueous solution A) and acetonitrile B), at a flow rate of 0.3 ml minhttp://www.swisstargetprediction.ch/) (https://www.genecards.org/) and MalaCards (http://www.uniprot.org/). Then, the overlapping targets of constituents and liver diseases were screened out as potential targets. To investigate their interactions, all potential targets were uploaded into the STRING database. A protein-protein interaction (PPI) network was constructed by Cytoscape 3.7.1 software and topological analysis of nodes in the network was performed (https://david.ncifcrf.gov/). In the end, a comprehensive network of “TCM formula-botanical drugs-compounds-targets-pathways” against liver diseases was established by Cytoscape 3.7.1 software.First of all, 11 secondary metabolites with specificity and measurability detected in serum and their 15 metabolites see  were selion.ch/) . Two pubalaCards , were aperformed . GO funcUnder optimized LC and MS conditions, the UPLC-Q-Exactive Orbitrap/MS technology was adopted to rapidly identify secondary metabolites of HGT as much as possible. However, for the research of TCM formulas, there was a key challenge if the chemical components were identified directly, the high response peaks might cover the small response peaks, resulting in the difficulty of identification. Hence, secondary metabolites of HGT were characterized systematically using the “representative secondary metabolites-botanical drugs-TCM formula” strategy, which was proposed in our previous work . At firsn data of the identified chemical component in HGT and its botanical drugs were listed in As illustrated in , indicating that the established method was feasible and suitable for the quality evaluation of HGT.Under the optimal conditions, the precision, stability, repeatability as well as durability of the method were evaluated with the reference peaks of SS2 (250 nm) and SSDA (200 nm), which contribute to establishing the HPLC characteristic chromatogram. The detailed results were displayed in . In conclusion, this study clarified 26 characteristic secondary metabolites, and provided a basis for the discovery of the Q-markers in the following tests.The validated HPLC method was applied to analyze 15 batches of HGT. The results validated that the similarity of the 15 batches of HGT was greater than 0.97, which conformed to the requirements of ChP. As shown in . In conAs shown in r values were above 0.9998) in a limited linear range. And the corresponding RSD of precision and stability were less than 2.1% and 2.3%, respectively. Then, the RSD of repeatability variations ranged from 2.2% to 3.8%. Moreover, the recovery of index compounds was proved to be satisfactory as they ranged from 95.10% to 103.99% with RSD of 2.3%–8.1%. Based on the above method, the contents of 11 specific compounds in 15 batches of HGT were calculated, and the results were displayed in In quality control of TCM preparations, the core step is to establish the quantitative analysis method of index components, because the measurability is also a basic requirement of the Q-markers . Consequin vivo level, and three secondary metabolites containing -goitrin, TA and GA, inhibited liver disease-related indicator (inflammatory factors) at the in vitro level. The detailed literature mining method and results were presented in in vivo biotransformation studies on these 11 secondary metabolites to obtain their in vivo biotransformation forms for network analysis.Based on the above experimental results, 11 plant metabolites in HGT met both the measurability and specificity requirements for Q-markers. In addition, the detailed literature investigation of these 11 plant metabolites was conducted. We found that Schisandra Transaminase reduction capsule with SSD, SSDA and SSDB as the main ingredients has the effects of astringency, regulating qi and transaminase reduction . And thein vivo, eight secondary metabolites were screened out as the representative secondary metabolites of HGT to investigate their typical biotransformation pathways in vivo, which contributed to the biotransformation profile analysis in vivo of HGT as a complicated system. Therefore, a new strategy of “from monomer to TCM formula, from high dose to clinical equivalent dose” was introduced to identify effective ingredients in vivo of TCM formula and provide chemical compounds basis for the next network analysis.After oral administration, TCM formulas, through the synergetic effects of the digestive tract and gastrointestinal flora, produce mixtures consisting of the inherent components and their metabolites, some of which are excreted directly, while others are selectively absorbed and enter the bloodstream under the assistance of liver metabolizing enzymes to exert medicinal effects . Admitten data of 26 components were summarized in in vivo, namely, oxidation, demethylation, methylation, methylenedioxy ring opening as well as hydroxylation, whose biotransformation pathway was displayed in Based on the strategy of “from monomer to TCM formula, from high dose to clinical equivalent dose” strategy, 11 prototypical secondary metabolites and 15 metabolites derived from HGT were identified in rats after oral administration of clinical equivalent dose groups , suggest7) was observed in the extracted ion chromatogram (EIC) of a representative compound group (5) in the clinical equivalent dose of HGT group, leading to its fragment ions with small response values were disturbed by other fragment ions -goitrin by the comparison with MSn data and retention time of the reference standard. Taken together, our proposed method can not only effectively identify low-response metabolites, but also enhance the reliability of metabolite identification in serum.Taking the identification of a metabolite as an example, the F2 peak with a higher response -goitrin, SSTA, SSDC, SSDA and GA, could exert their effects directly in vivo in the form of prototype components. Therefore, integrating network analysis and biotransformation pathway research of specific secondary metabolites, 9 secondary metabolites were identified as Q-markers of HGT: SS2 in BR, -goitrin in IR, CA in ASH, SSD, SZDB, SSTA, SSDA, SSDC in SCF and GA in PFS.A comprehensive network of “TCM formula-botanical drugs-components-targets-pathways” was built to reveal the complex interactions, and screen out Q-marker candidates of HGT. Making use of the corresponding database, 597 potential targets of 26 components in serum were obtained, and 1762 liver diseases-related human targets were collected. Subsequently, the targets of the compounds intersected with the disease targets to form 213 overlapping targets , and a Ppathways , includipathways , which cin vivo or in vitro levels, which provided clear literature support for our study and demonstrates the reliability of our research results to a certain extent. In view of the above views, 9 Q-markers of HGT identified by the funnel-type stepwise filtering strategy possess comprehensive features of compatibility, quality transitivity and traceability, specificity, measurability and effectiveness to some extent. Whereas, there are some limitations in this study, such as the effectiveness of Q-markers has yet to be confirmed on the basis of in vivo and in vitro experiments.Here, a funnel-type stepwise filtering strategy integrating secondary metabolites characterization, characteristic chromatogram, quantitative analysis, literature mining, biotransformation pathways and network analysis was successfully developed and applied to screen out 9 representative Q-markers of HGT. Admittedly, we currently only speculated that these 9 Q-markers might be active ingredients of HGT through network analysis. However, as mentioned above, relevant studies reported that 9 Q-markers had therapeutic effects on liver disease at the Noteworthy, our study not only systematically identified Q-markers of HGT, but also conducted an exploratory study on the mechanism for the treatment of liver diseases based on network analysis. Based on the integrated network, 9 Q-markers may regulate phosphatidylinositol-3-kinase/Akt (PI3K-Akt), FoxO, tumor necrosis factor (TNF), mitogen-activated protein kinase (MAPK), HIF-1, vascular endothelial growth factor (VEGF) and other signaling pathways through acting on AKT1, TNF, EGFR, JUN, STAT3, SRC, CASP3, MAPK3, HSP90AA1 and HIF1A targets to achieve the protective effect on the liver. Oxidative stress has been closely associated with almost all human liver diseases . The rel


Neurofibromatosis type 2 (NF-2) is a genetic condition that by definition includes bilateral vestibular schwannoma, a non-malignant lesion also known as acoustic neuroma. Patients often develop hearing impairment and hearing loss as a result of the involvement of the vestibulocochlear nerve bilaterally as well as attempts at surgical repair. A common treatment for NF-2-mediated schwannoma is the antiangiogenic agent, bevacizumab. In many cases, patients require prolonged and even lifelong treatment with bevacizumab to control schwannoma growth. However, long-term use of bevacizumab can be associated with multiple side effects including hypertension and proteinuria including nephrotic syndrome (>3g of protein excreted in the urine in 24 hours). In these situations, the challenge with discontinuing prolonged bevacizumab can be rapid tumor growth and worsening hearing loss. Pre-clinical data suggests that hearing loss can be prevented following treatment with the angiotensin receptor blocker (ARB), losartan, in an animal model of NF-2. ARBs are already established in nephrology guidelines to treat proteinuria and hypertension. Here, we present a patient with NF-2 who developed nephrotic syndrome while on bevacizumab. Attempts to discontinue bevacizumab resulted in near-immediate hearing loss. Treatment with the ARB telmisartan together with bevacizumab resulted in improved hearing, reduced proteinuria, and controlled hypertension.  Neurofibromatosis type 2 (NF-2) is an autosomal dominant disorder resulting in the eventual development of several types of benign neoplasms within the nervous system. The development of bilateral vestibular schwannoma is so common that it is part of the major diagnostic criteria. While the tumors are benign, their predilection for growing in the vestibulocochlear nerve results in a decreased quality of life for patients because of hearing impairment. Bevacizumab has been used to improve hearing loss and manage tumor growth with clinical trial data demonstrating efficacy in doing so. A consequence of long-term bevacizumab use is nephrotic syndrome with proteinuria. Angiotensin receptor blockade concomitant with bevacizumab may have a synergistic therapeutic effect as seen in metastatic colorectal cancer. This case report illustrates that angiotensin receptor blockade concurrent with bevacizumab can reduce bevacizumab-induced proteinuria and can subjectively improve hearing loss. This hypothesis-generating case report supports the use of angiotensin receptor blockers (ARB) in NF-2 patients treated with long-term bevacizumab. It provides a rationale to investigate the timing of ARB use in NF-2 patients with long-term bevacizumab use given that one-third of patients will develop hypertension and proteinuria and that most patients will have hearing impairment. A 38-year-old female previously diagnosed with NF-2 presented with chronic, progressive hearing loss and postural imbalance. She had bilateral vestibular schwannoma as well as supratentorial and spinal column meningioma. She had a translabyrinthine craniotomy and resection of vestibular schwannoma and instrumented cranial vault repair on the right side on July 10, 2017. This resulted in complete right-sided hearing loss. She was symptomatic in the left ear with significant tinnitus as well as subjective hearing loss. She was offered bevacizumab to preserve and maintain hearing in the left ear. Bevacizumab was given intravenously at a dose of 5 mg/kg of body weight every two weeks. Her blood pressure prior to starting treatment was 104/68 mmHg. At the time of writing, the patient had received 124 cycles of bevacizumab.Within seven months of treatment, her tumor volumes decreased Figure  and she NF-2 is a heritable disorder resulting in the eventual development of different tumor types affecting the nervous system including schwannoma, meningioma, and ependymoma.. Patients characteristically develop at least unilateral vestibular schwannoma, formerly known as acoustic neuroma, but commonly develop bilateral vestibular schwannoma. The development of bilateral vestibular schwannoma is one of the major diagnostic criteria for the disease and is present in 95% of cases. NF-2 has an incidence of one in 40,000 . It is aStandard therapy for growing sporadic, unilateral vestibular schwannomas includes surgical removal or radiation therapy. Both treatments usually achieve tumor control, but at the frequent cost of hearing loss in the affected ear . Only 41A particular challenge is the development of nephrotic range proteinuria. This occurs through several pathomechanisms but a principal etiology is glomerular microangiopathy. It has not been shown conclusively to be a dose-dependent side effect. The incidence of proteinuria with bevacizumab in one study (n=42) was 35.7% with a median time to onset of 213 days. In the same study, the incidence of hypertension was 52.% with a median time to hypertension of 84 days . ARB is This case report highlights the potential synergy with the concurrent use of ARB and bevacizumab in patients with NF-2. It suggests more research should be done to detail the mechanisms underlying tumor suppression and hearing improvement with this treatment strategy.