Creating a health informatics data resource for hearing health research.

BACKGROUND: The National Institute of Health and Social Care Research (NIHR) Health Informatics Collaborative (HIC) for Hearing Health has been established in the UK to curate routinely collected hearing health data to address research questions. This study defines priority research areas, outlines its aims, governance structure and demonstrates how hearing health data have been integrated into a common data model using pure tone audiometry (PTA) as a case study. METHODS: After identifying key research aims in hearing health, the governance structure for the NIHR HIC for Hearing Health is described. The Observational Medical Outcomes Partnership (OMOP) was chosen as our common data model to provide a case study example. RESULTS: The NIHR HIC Hearing Health theme have developed a data architecture outlying the flow of data from all of the various siloed electronic patient record systems to allow the effective linkage of data from electronic patient record systems to research systems. Using PTAs as an example, OMOPification of hearing health data successfully collated a rich breadth of datapoints across multiple centres. CONCLUSION: This study identified priority research areas where routinely collected hearing health data could be useful. It demonstrates integration and standardisation of such data into a common data model from multiple centres. By describing the process of data sharing across the HIC, we hope to invite more centres to contribute and utilise data to address research questions in hearing health. This national initiative has the power to transform UK hearing research and hearing care using routinely collected clinical data.

Acoustic reflexes: should we be paying more attention?

OBJECTIVE: The clinical audiology test battery often involves playing physically simple sounds with questionable ecological value to the listener. In this technical report, we revisit how valid this approach is using an automated, involuntary auditory response; the acoustic reflex threshold (ART). DESIGN: The ART was estimated four times in each individual in a quasi-random ordering of task conditions. The baseline condition (referred to as Neutral) measured the ART following a standard clinical practice. Three experimental conditions were then used in which a secondary task was performed whilst the reflex was measured: auditory attention, auditory distraction and visual distraction tasks. STUDY SAMPLE: Thirty-eight participants (27 males) with a mean age of 23 years were tested. All participants were audiometrically healthy. RESULTS: The ART was elevated when a visual task was performed at the same time as the measurements were taken. Performing an auditory task did not affect the ART. CONCLUSIONS: These data indicate that simple audiometric measures widely used in the clinic, can be affected by central, non-auditory processes even in healthy, normal-hearing volunteers. The role of cognition and attention on auditory responses will become ever more important in the coming years.

Cogmed Training Does Not Generalize to Real-World Benefits for Adult Hearing Aid Users: Results of a Blinded, Active-Controlled Randomized Trial.

OBJECTIVES: Performance on working memory tasks is positively associated with speech-in-noise perception performance, particularly where auditory inputs are degraded. It is suggested that interventions designed to improve working memory capacity may improve domain-general working memory performance for people with hearing loss, to benefit their real-world listening. We examined whether a 5-week training program that primarily targets the storage component of working memory (Cogmed RM, adaptive) could improve cognition, speech-in-noise perception and self-reported hearing in a randomized controlled trial of adult hearing aid users with mild to moderate hearing loss, compared with an active control (Cogmed RM, nonadaptive) group of adults from the same population. DESIGN: A preregistered randomized controlled trial of 57 adult hearing aid users (n = 27 experimental, n = 30 active control), recruited from a dedicated database of research volunteers, examined on-task learning and generalized improvements in measures of trained and untrained cognition, untrained speech-in-noise perception and self-reported hearing abilities, pre- to post-training. Participants and the outcome assessor were both blinded to intervention allocation. Retention of training-related improvements was examined at a 6-month follow-up assessment. RESULTS: Per-protocol analyses showed improvements in trained tasks (Cogmed Index Improvement) that transferred to improvements in a trained working memory task tested outside of the training software (Backward Digit Span) and a small improvement in self-reported hearing ability (Glasgow Hearing Aid Benefit Profile, Initial Disability subscale). Both of these improvements were maintained 6-month post-training. There was no transfer of learning shown to untrained measures of cognition (working memory or attention), speech-in-noise perception, or self-reported hearing in everyday life. An assessment of individual differences showed that participants with better baseline working memory performance achieved greater learning on the trained tasks. Post-training performance for untrained outcomes was largely predicted by individuals' pretraining performance on those measures. CONCLUSIONS: Despite significant on-task learning, generalized improvements of working memory training in this trial were limited to (a) improvements for a trained working memory task tested outside of the training software and (b) a small improvement in self-reported hearing ability for those in the experimental group, compared with active controls. We found no evidence to suggest that training which primarily targets storage aspects of working memory can result in domain-general improvements that benefit everyday communication for adult hearing aid users. These findings are consistent with a significant body of evidence showing that Cogmed training only improves performance for tasks that resemble Cogmed training. Future research should focus on the benefits of interventions that enhance cognition in the context in which it is employed within everyday communication, such as training that targets dynamic aspects of cognitive control important for successful speech-in-noise perception.

Velvet domain protein VosA represses the zinc cluster transcription factor SclB regulatory network for Aspergillus nidulans asexual development, oxidative stress response and secondary metabolism.

The NF-κB-like velvet domain protein VosA (viability of spores) binds to more than 1,500 promoter sequences in the filamentous fungus Aspergillus nidulans. VosA inhibits premature induction of the developmental activator gene brlA, which promotes asexual spore formation in response to environmental cues as light. VosA represses a novel genetic network controlled by the sclB gene. SclB function is antagonistic to VosA, because it induces the expression of early activator genes of asexual differentiation as flbC and flbD as well as brlA. The SclB controlled network promotes asexual development and spore viability, but is independent of the fungal light control. SclB interactions with the RcoA transcriptional repressor subunit suggest additional inhibitory functions on transcription. SclB links asexual spore formation to the synthesis of secondary metabolites including emericellamides, austinol as well as dehydroaustinol and activates the oxidative stress response of the fungus. The fungal VosA-SclB regulatory system of transcription includes a VosA control of the sclB promoter, common and opposite VosA and SclB control functions of fungal development and several additional regulatory genes. The relationship between VosA and SclB illustrates the presence of a convoluted surveillance apparatus of transcriptional control, which is required for accurate fungal development and the linkage to the appropriate secondary metabolism.

Correction: Velvet domain protein VosA represses the zinc cluster transcription factor SclB regulatory network for Aspergillus nidulans asexual development, oxidative stress response and secondary metabolism.

[This corrects the article DOI: 10.1371/journal.pgen.1007511.].

Financial reward has differential effects on behavioural and self-report measures of listening effort.

OBJECTIVES: To investigate the effects of listening demands and motivation on listening effort (LE) in a novel speech recognition task. DESIGN: We manipulated listening demands and motivation using vocoded speech and financial reward, respectively, and measured task performance (correct response rate) and indices of LE (response times (RTs), subjective ratings of LE and likelihood of giving up). Effects of inter-individual differences in cognitive skills and personality on task performance and LE were also assessed within the context of the Cognitive Energetics Theory (CET). STUDY SAMPLE: Twenty-four participants with normal-hearing (age range: 19 - 33 years, 6 male). RESULTS: High listening demands decreased the correct response rate and increased RTs, self-rated LE and self-rated likelihood of giving up. High financial reward increased subjective LE ratings only. Mixed-effects modelling showed small fixed effects for competitiveness on LE measured using RTs. Small fixed effects were found for cognitive skills (lexical decision RTs and backwards digit span) on LE measured using RTs and correct response rate, respectively. CONCLUSIONS: The effects of listening demands on LE in the speech recognition task aligned with CET, whereas predictions regarding the influence of motivation, cognitive skills and personality were only partially supported.

Effects of Cognitive Load on Pure-Tone Audiometry Thresholds in Younger and Older Adults.

OBJECTIVES: Cognitive load (CL) impairs listeners' ability to comprehend sentences, recognize words, and identify speech sounds. Recent findings suggest that this effect originates in a disruption of low-level perception of acoustic details. Here, we attempted to quantify such a disruption by measuring the effect of CL (a two-back task) on pure-tone audiometry (PTA) thresholds. We also asked whether the effect of CL on PTA was greater in older adults, on account of their reduced ability to divide cognitive resources between simultaneous tasks. To specify the mechanisms and representations underlying the interface between auditory and cognitive processes, we contrasted CL requiring visual encoding with CL requiring auditory encoding. Finally, the link between the cost of performing PTA under CL, working memory, and speech-in-noise (SiN) perception was investigated and compared between younger and older participants. DESIGN: Younger and older adults (44 in each group) did a PTA test at 0.5, 1, 2, and 4 kHz pure tones under CL and no CL. CL consisted of a visual two-back task running throughout the PTA test. The two-back task involved either visual encoding of the stimuli (meaningless images) or subvocal auditory encoding (a rhyme task on written nonwords). Participants also underwent a battery of SiN tests and a working memory test (letter number sequencing). RESULTS: Younger adults showed elevated PTA thresholds under CL, but only when CL involved subvocal auditory encoding. CL had no effect when it involved purely visual encoding. In contrast, older adults showed elevated thresholds under both types of CL. When present, the PTA CL cost was broadly comparable in younger and older adults (approximately 2 dB HL). The magnitude of PTA CL cost did not correlate significantly with SiN perception or working memory in either age group. In contrast, PTA alone showed strong links to both SiN and letter number sequencing in older adults. CONCLUSIONS: The results show that CL can exert its effect at the level of hearing sensitivity. However, in younger adults, this effect is only found when CL involves auditory mental representations. When CL involves visual representations, it has virtually no impact on hearing thresholds. In older adults, interference is found in both conditions. The results suggest that hearing progresses from engaging primarily modality-specific cognition in early adulthood to engaging cognition in a more undifferentiated way in older age. Moreover, hearing thresholds measured under CL did not predict SiN perception more accurately than standard PTA thresholds.

Hearing in Real-Life Environments (HERE): Structure and Reliability of a Questionnaire on Perceived Hearing for Older Adults.

OBJECTIVES: The ability to hear in a variety of social situations and environments is vital for social participation and a high quality of life. One way to assess hearing ability is by means of self-report questionnaire. For questionnaires to be useful, their measurement properties, based on careful validation, have to be known. Only recently has consensus been reached concerning how to perform such validation and been published as COSMIN (consensus-based standards for the selection of health status measurement instruments) guidelines. Here the authors use these guidelines to evaluate the measurement properties of the "Hearing in Real-Life Environments" (HERE) questionnaire, a newly developed self-report measure that assesses speech perception, spatial orientation, and the social-emotional consequences of hearing impairment in older adults. The aim is to illustrate the process of validation and encourage similar examinations of other frequently used questionnaires. DESIGN: The HERE questionnaire includes 15 items with a numeric rating scale from 0 to 10 for each item and allows the assessment of hearing with and without hearing aids. The evaluation was performed in two cohorts of community-dwelling older adults from Finland (n = 581, mean 82 years) and the United Kingdom (n = 50, mean 69 years). The internal structure of the questionnaire and its relationship to age, hearing level, and self-reported and behavioral measures of speech perception was assessed and, when possible, compared between cohorts. RESULTS: The results of the factor analysis showed that the HERE's internal structure was similar across cohorts. In both cohorts, the factor analysis showed a satisfactory solution for three factors (speech hearing, spatial hearing, and socio-emotional consequences), with a high internal consistency for each factor (Cronbach's α's for the factors from 0.90 to 0.97). Test-retest analysis showed the HERE overall mean score to be stable and highly replicable over time (intraclass correlation coefficient = 0.86, standard error of measurement of the test score = 0.92). The HERE overall mean score correlated highly with another self-report measure of speech perception, the Speech Spatial Qualities of Hearing questionnaire (standardized regression coefficient [ß] = -0.75, p < 0.001), moderately highly with behaviorally assessed hearing level (best-ear average: ß = 0.45 to 0.46), and moderately highly with behaviorally measured intelligibility of sentences in noise (ß = -0.50, p < 0.001). CONCLUSIONS: Using the COSMIN guidelines, the authors show that the HERE is a valid, reliable, and stable questionnaire for the assessment of self-reported speech perception, sound localization, and the socio-emotional consequences of hearing impairment in the context of social functioning. The authors also show that cross-cultural data collected using different data collection strategies can be combined with a range of statistical methods to validate a questionnaire.

Promoter Activation in Δhfq Mutants as an Efficient Tool for Specialized Metabolite Production Enabling Direct Bioactivity Testing.

Natural products (NPs) from microorganisms have been important sources for discovering new therapeutic and chemical entities. While their corresponding biosynthetic gene clusters (BGCs) can be easily identified by gene-sequence-similarity-based bioinformatics strategies, the actual access to these NPs for structure elucidation and bioactivity testing remains difficult. Deletion of the gene encoding the RNA chaperone, Hfq, results in strains losing the production of most NPs. By exchanging the native promoter of a desired BGC against an inducible promoter in Δhfq mutants, almost exclusive production of the corresponding NP from the targeted BGC in Photorhabdus, Xenorhabdus and Pseudomonas was observed including the production of several new NPs derived from previously uncharacterized non-ribosomal peptide synthetases (NRPS). This easyPACId approach (easy Promoter Activated Compound Identification) facilitates NP identification due to low interference from other NPs. Moreover, it allows direct bioactivity testing of supernatants containing secreted NPs, without laborious purification.

Natural Products from Photorhabdus and Other Entomopathogenic Bacteria.

Although the first natural products (NP) from Photorhabdus and Xenorhabdus bacteria have been known now for almost 30 years, a huge variety of new compounds have been identified in the last 5-10 years, mainly due to the application of modern mass spectrometry. Additionally, application of molecular methods that allow the activation of NP production in several different strains as well as efficient heterologous expression methods have led to the production and validation of many new compounds. In this chapter we discuss the benefit of using Photorhabdus as a model system for microbial chemical ecology. We also examine non-ribosomal peptide synthetases as the most important pathway for NP production. Finally, we discuss the origin and function of all currently known NPs and the development of the molecular and chemical tools used to identify these NPs faster.

Photorhabdus-nematode symbiosis is dependent on hfq-mediated regulation of secondary metabolites.

Photorhabdus luminescens maintains a symbiotic relationship with the nematodes Heterorhabditis bacteriophora and together they infect and kill insect larvae. To maintain this symbiotic relationship, the bacteria must produce an array of secondary metabolites to assist in the development and replication of nematodes. The regulatory mechanisms surrounding production of these compounds are mostly unknown. The global post-transcriptional regulator, Hfq, is widespread in bacteria and performs many functions, one of which is the facilitation of sRNA binding to target mRNAs, with recent research thoroughly exploring its various pleiotropic effects. Here we generate and characterize an hfq deletion mutant and show that in the absence of hfq, the bacteria are no longer able to maintain a healthy symbiosis with nematodes due to the abolishment of the production of all known secondary metabolites. RNAseq led us to produce a second deletion of a known repressor, HexA, in the same strain, which restored both metabolite production and symbiosis.

The Contribution of Auditory and Cognitive Factors to Intelligibility of Words and Sentences in Noise.

Understanding the causes for speech-in-noise (SiN) perception difficulties is complex, and is made even more difficult by the fact that listening situations can vary widely in target and background sounds. While there is general agreement that both auditory and cognitive factors are important, their exact relationship to SiN perception across various listening situations remains unclear. This study manipulated the characteristics of the listening situation in two ways: first, target stimuli were either isolated words, or words heard in the context of low- (LP) and high-predictability (HP) sentences; second, the background sound, speech-modulated noise, was presented at two signal-to-noise ratios. Speech intelligibility was measured for 30 older listeners (aged 62-84) with age-normal hearing and related to individual differences in cognition (working memory, inhibition and linguistic skills) and hearing (PTA(0.25-8 kHz) and temporal processing). The results showed that while the effect of hearing thresholds on intelligibility was rather uniform, the influence of cognitive abilities was more specific to a certain listening situation. By revealing a complex picture of relationships between intelligibility and cognition, these results may help us understand some of the inconsistencies in the literature as regards cognitive contributions to speech perception.

A Photorhabdus natural product inhibits insect juvenile hormone epoxide hydrolase.

Simple urea compounds ("phurealipids") have been identified from the entomopathogenic bacterium Photorhabdus luminescens, and their biosynthesis was elucidated. Very similar analogues of these compounds have been previously developed as inhibitors of juvenile hormone epoxide hydrolase (JHEH), a key enzyme in insect development and growth. Phurealipids also inhibit JHEH, and therefore phurealipids might contribute to bacterial virulence.

Influences of word predictability and type of masker noise on intelligibility of sung text in live concerts.

Vocal music is often intended to convey meaning, but how effectively this is achieved is poorly understood. This study systematically assessed the influence of three non-phonetic factors on the intelligibility of sung words in six public concerts in different venues: word predictability from sentence context, type of masker noise (spoken babble, sung vowels, [∫(w)]), and signal-to-noise ratio (SNR). Stimuli were sung live by a professional a cappella ensemble with one male singing target sentences and five others (two female) producing the masker sounds. The concert audiences (N = 319) reported the final word of each sentence using a handheld voting device, from four phonetically- and semantically-controlled written alternatives projected onto a screen after the sentence was sung. Although overall accuracy differed between performances, intelligibility patterns were robust across concerts. They included predicted main effects of masker noise type ([∫(w)] masking least disruptive, babble most), SNR (high > low), semantic predictability (high > low), listener age (young > old), and listener language status (native > non-native), and some strong interactions. These results suggest that, despite acoustic differences between sung and spoken words and the unusual and varied experimental venues, key findings from traditional speech research apply to sung words, given appropriate musical composition.

Divergent effects of listening demands and evaluative threat on listening effort in online and laboratory settings.

Objective: Listening effort (LE) varies as a function of listening demands, motivation and resource availability, among other things. Motivation is posited to have a greater influence on listening effort under high, compared to low, listening demands. Methods: To test this prediction, we manipulated the listening demands of a speech recognition task using tone vocoders to create moderate and high listening demand conditions. We manipulated motivation using evaluative threat, i.e., informing participants that they must reach a particular "score" for their results to be usable. Resource availability was assessed by means of working memory span and included as a fixed effects predictor. Outcome measures were indices of LE, including reaction times (RTs), self-rated work and self-rated tiredness, in addition to task performance (correct response rates). Given the recent popularity of online studies, we also wanted to examine the effect of experimental context (online vs. laboratory) on the efficacy of manipulations of listening demands and motivation. We carried out two highly similar experiments with two groups of 37 young adults, a laboratory experiment and an online experiment. To make listening demands comparable between the two studies, vocoder settings had to differ. All results were analysed using linear mixed models. Results: Results showed that under laboratory conditions, listening demands affected all outcomes, with significantly lower correct response rates, slower RTs and greater self-rated work with higher listening demands. In the online study, listening demands only affected RTs. In addition, motivation affected self-rated work. Resource availability was only a significant predictor for RTs in the online study. Discussion: These results show that the influence of motivation and listening demands on LE depends on the type of outcome measures used and the experimental context. It may also depend on the exact vocoder settings. A controlled laboratory settings and/or particular vocoder settings may be necessary to observe all expected effects of listening demands and motivation.

A review of auditory processing and cognitive change during normal ageing, and the implications for setting hearing aids for older adults.

Throughout our adult lives there is a decline in peripheral hearing, auditory processing and elements of cognition that support listening ability. Audiometry provides no information about the status of auditory processing and cognition, and older adults often struggle with complex listening situations, such as speech in noise perception, even if their peripheral hearing appears normal. Hearing aids can address some aspects of peripheral hearing impairment and improve signal-to-noise ratios. However, they cannot directly enhance central processes and may introduce distortion to sound that might act to undermine listening ability. This review paper highlights the need to consider the distortion introduced by hearing aids, specifically when considering normally-ageing older adults. We focus on patients with age-related hearing loss because they represent the vast majority of the population attending audiology clinics. We believe that it is important to recognize that the combination of peripheral and central, auditory and cognitive decline make older adults some of the most complex patients seen in audiology services, so they should not be treated as "standard" despite the high prevalence of age-related hearing loss. We argue that a primary concern should be to avoid hearing aid settings that introduce distortion to speech envelope cues, which is not a new concept. The primary cause of distortion is the speed and range of change to hearing aid amplification (i.e., compression). We argue that slow-acting compression should be considered as a default for some users and that other advanced features should be reconsidered as they may also introduce distortion that some users may not be able to tolerate. We discuss how this can be incorporated into a pragmatic approach to hearing aid fitting that does not require increased loading on audiology services.

Sex differences and the effect of female sex hormones on auditory function: a systematic review.

Aims: First, to discuss sex differences in auditory function between women and men, and whether cyclic fluctuations in levels of female sex hormones (i.e., estradiol and progesterone) affect auditory function in pre-menopausal and post-menopausal women. Second, to systematically review the literature concerning the discussed patterns in order to give an overview of the methodologies used in research. Last, to identify the gap in knowledge and to make recommendations for future work. Methods for the systematic review: Population, Exposure, Control, Outcome and Study design (PECOS) criteria were used in developing the review questions. The review protocol follows the guidelines of the Preferred Reporting Items for Systematic Reviews and Meta-Analyses (PRISMA) and was pre-registered in the Prospective Register of Systematic Reviews (PROSPERO; CRD42020201480). Data Sources: EMBASE, PubMed, MEDLINE (Ovid), PsycINFO, ComDisDome, CINAHL, Web of Science, Cochrane Central Register of Controlled Trials (CENTRAL) via Cochrane Library, and scanning reference lists of relevant studies, and internet resources (i.e., Mendeley) were used. Only studies published between 1999 and 2022, in English, or in English translation, were included. The quality of evidence was assessed using the Newcastle-Ottawa Scale (NOS). Results: Sex differences: Women had more sensitive hearing (measured at the level of peripheral and central auditory system) than men. Cyclic fluctuations: Auditory function in women fluctuated during the menstrual cycle, while no such fluctuations in men over the same time period were reported. Hearing sensitivity improved in women during the late follicular phase, and decrease during the luteal phase, implying an effect of female sex hormones, although the specific effects of estradiol and progesterone fluctuations on the central auditory system remain unclear. Hearing sensitivity in women declined rapidly at the onset of menopause. Conclusion: The review has shown the following. Consistent sex differences exist in auditory function across the auditory pathway with pre-menopausal women often showing better function than age-matched men. Moreover, pre-menopausal women show fluctuations in hearing function across the menstrual cycle with a better function during the peak of estradiol or when the ratio of estradiol to progesterone is high. Third, menopause marks the onset of hearing loss in women, characterized by a rapid decline in hearing sensitivity and a more pronounced loss than in age-matched men. Finally, the systematic review highlights the need for well-designed and -controlled studies to evaluate the influence of estradiol and progesterone on hearing by consistently including control groups (e.g., age-matched man), using objective tests to measure hormonal levels (e.g., in saliva or blood), and by testing participants at different points across the menstrual cycle. Systematic review registration: https://www.crd.york.ac.uk/prospero/display_record.php?ID=CRD42020201480, identifier CRD42020201480.

Relationship between self-reported listening and communication difficulties and executive function: a protocol for a systematic review and meta-analysis.

INTRODUCTION: Listening and communication difficulties can limit people's participation in activity and adversely affect their quality of life. Hearing, as well as listening and communication difficulties, can be measured either by using behavioural tests or self-report measures, and the outcomes are not always closely linked. The association between behaviourally measured and self-reported hearing is strong, whereas the association between behavioural and self-reported measures of listening and communication difficulties is much weaker, suggesting they assess different aspects of listening. While behavioural measures of listening and communication difficulties have been associated with poorer cognitive performance including executive functions, the same association has not always been shown for self-report measures. The objective of this systematic review and meta-analysis is to understand the relationship between executive function and self-reported listening and communication difficulties in adults with hearing loss, and where possible, potential covariates of age and pure-tone audiometric thresholds. METHODS AND ANALYSIS: Studies will be eligible for inclusion if they report data from both a self-report measure of listening difficulties and a behavioural measure of executive function. Eight databases are to be searched: MEDLINE (via Ovid SP), EMBASE (via Ovid SP), PsycINFO (via Ovid SP), ASSIA (via ProQuest), Cumulative Index to Nursing and Allied Health Literature or CINAHL (via EBSCO Host), Scopus, PubMed and Web of Science (Science and Social Science Citation Index). The JBI critical appraisal tool will be used to assess risk of bias for included studies. Results will be synthesised primarily using a meta-analysis, and where sufficient quantitative data are not available, a narrative synthesis will be carried out to describe key results. ETHICS AND DISSEMINATION: No ethical issues are foreseen. Data will be disseminated via academic publication and conference presentations. Findings may also be published in scientific newsletters and magazines. PROSPERO REGISTRATION NUMBER: CRD42022293546.

A set of closely related methyltransferases for site-specific tailoring of anthraquinone pigments.

Modification of the polyketide anthraquinone AQ-256 in the entomopathogenic Photorhabdus luminescens involves several O-methylations, but the biosynthetic gene cluster antA-I lacks corresponding tailoring enzymes. We here describe the identification of five putative, highly homologous O-methyltransferases encoded in the genome of P. luminescens. Activity assays in vitro and deletion experiments in vivo revealed that three of them account for anthraquinone tailoring by producing three monomethylated and two dimethylated species of AQ-256. X-ray structures of all five enzymes indicate high structural and mechanistic similarity. As confirmed by structure-based mutagenesis, a conserved histidine at the active site likely functions as a general base for substrate deprotonation and subsequent methyl transfer in all enzymes. Eight complex structures with AQ-256 as well as mono- and dimethylated derivatives confirm the substrate specificity patterns found in vitro and visualize how single amino acid differences in the active-site pockets impact substrate orientation and govern site-specific methylation.