Examination of recent hydroelectric dam projects in Canada for alignment of baseline studies, predictive modeling, and postdevelopment monitoring phases of aquatic environmental impact assessments.

Environmental impact assessment (EIA) has been widely criticized by the aquatic science community for poorly aligned approaches when selecting endpoints and collecting data during the baseline, predictive modeling, and postdevelopment monitoring phases. If these critical phases of the EIA process are not aligned properly, it can be difficult to evaluate the presence of postdevelopment effects. Examples of the misalignment of these phases include baseline studies failing to measure indicators that are monitored postdevelopment; predictive assessments that do not quantitatively predict conditions or potential impacts postdevelopment; and the failure to identify relevant indicators that may detect effects postdevelopment. For aquatic assessments, understanding how to protect critical ecosystem attributes to satisfy regulatory concerns could help to better align aquatic science monitoring activities across EIA phases. In this article we investigate recent Canadian hydroelectric dam EIAs to evaluate how well recent assessment approaches are meeting these necessary conditions of good aquatic EIA practice through the lens of ecosystem services from a fish's perspective. We found that larger facilities generally had baseline studies and modeling that better supported postdevelopment monitoring, but improvements in structure, linkages, and expectations would better align EIA phases in a manner that would improve assessments and environmental protection. Integr Environ Assess Manag 2024;20:616-644. © 2023 The Authors. Integrated Environmental Assessment and Management published by Wiley Periodicals LLC on behalf of Society of Environmental Toxicology & Chemistry (SETAC).

Out of the Silence: Insights into How Genes Escape X-Chromosome Inactivation.

The silencing of all but one X chromosome in mammalian cells is a remarkable epigenetic process leading to near dosage equivalence in X-linked gene products between the sexes. However, equally remarkable is the ability of a subset of genes to continue to be expressed from the otherwise inactive X chromosome-in some cases constitutively, while other genes are variable between individuals, tissues or cells. In this review we discuss the advantages and disadvantages of the approaches that have been used to identify escapees. The identity of escapees provides important clues to mechanisms underlying escape from XCI, an arena of study now moving from correlation to functional studies. As most escapees show greater expression in females, the not-so-inactive X chromosome is a substantial contributor to sex differences in humans, and we highlight some examples of such impact.

Refining the genomic determinants underlying escape from X-chromosome inactivation.

X-chromosome inactivation (XCI) epigenetically silences one X chromosome in every cell in female mammals. Although the majority of X-linked genes are silenced, in humans 20% or more are able to escape inactivation and continue to be expressed. Such escape genes are important contributors to sex differences in gene expression, and may impact the phenotypes of X aneuploidies; yet the mechanisms regulating escape from XCI are not understood. We have performed an enrichment analysis of transcription factor binding on the X chromosome, providing new evidence for enriched factors at the transcription start sites of escape genes. The top escape-enriched transcription factors were detected at the RPS4X promoter, a well-described human escape gene previously demonstrated to escape from XCI in a transgenic mouse model. Using a cell line model system that allows for targeted integration and inactivation of transgenes on the mouse X chromosome, we further assessed combinations of RPS4X promoter and genic elements for their ability to drive escape from XCI. We identified a small transgenic construct of only 6 kb capable of robust escape from XCI, establishing that gene-proximal elements are sufficient to permit escape, and highlighting the additive effect of multiple elements that work together in a context-specific fashion.

Acoustically Evoked Compound Action Potentials Recorded From Cochlear Implant Users With Preserved Acoustic Hearing.

OBJECTIVES: Less traumatic intracochlear electrode design and the introduction of the soft surgery technique allow for the preservation of low-frequency acoustic hearing in many cochlear implant (CI) users. Recently, new electrophysiologic methods have also been developed that allow acoustically evoked peripheral responses to be measured in vivo from an intracochlear electrode. These recordings provide clues to the status of peripheral auditory structures. Unfortunately, responses generated from the auditory nerve (auditory nerve neurophonic [ANN]) are somewhat difficult to record because they are smaller than the hair cell responses (cochlear microphonic). Additionally, it is difficult to completely segregate the ANN from the cochlear microphonic, complicating the interpretation and limiting clinical applications. The compound action potential (CAP) is a synchronous response of multiple auditory nerve fibers and may provide an alternative to ANN where the status of the auditory nerve is of primary interest. This study is a within-subject comparison of CAPs recorded using traditional stimuli (clicks and 500 Hz tone bursts) and a new stimulus (CAP chirp). We hypothesized that the chirp stimulus might result in a more robust CAP than that recorded using traditional stimuli, allowing for a more accurate assessment of the status of the auditory nerve. DESIGN: Nineteen adult Nucleus L24 Hybrid CI users with residual low-frequency hearing participated in this study. CAP responses were recorded from the most apical intracochlear electrode using a 100 µs click, 500 Hz tone bursts, and chirp stimuli presented via the insert phone to the implanted ear. The chirp stimulus used in this study was CAP chirp generated using parameters from human-derived band CAPs ( Chertoff et al. 2010 ). Additionally, nine custom chirps were created by systematically varying the frequency sweep rate of the power function used to construct the standard CAP chirp stimulus. CAPs were recorded using all acoustic stimuli, allowing for within-subject comparisons of the CAP amplitude, threshold, percentage of measurable CAP responses, and waveform morphology. RESULTS: Considerable variation in response morphology was apparent across stimuli and stimulation levels. Clicks and CAP chirp significantly evoked identifiable CAP response more compared to 500 Hz tone bursts. At relatively high stimulation levels, the chirp-evoked CAPs were significantly larger in amplitude and less ambiguous in morphology than the click-evoked CAPs. The status of residual acoustic hearing at high frequencies influenced the likelihood that a CAP could be reliably recorded. Subjects with better preserved hearing at high frequencies had significantly larger CAP amplitudes when CAP chirp was used. Customizing the chirp stimulus by varying the frequency sweep rates significantly affected the CAP amplitudes; however, pairwise comparisons did not show significant differences between chirps. CONCLUSIONS: CAPs can be measured more effectively using broadband acoustic stimuli than 500 Hz tone bursts in CI users with residual low-frequency acoustic hearing. The advantage of using CAP chirp stimulus relative to standard clicks is dependent on the extent of preserved acoustic hearing at high frequencies and the stimulus level. The chirp stimulus may present an attractive alternative to standard clicks or tone bursts for this CI population when the goal is to record robust CAP responses.

Longitudinal Electrocochleography as an Objective Measure of Serial Behavioral Audiometry in Electro-Acoustic Stimulation Patients.

OBJECTIVE: Minimally traumatic surgical techniques and advances in cochlear implant (CI) electrode array designs have allowed acoustic hearing present in a CI candidate prior to surgery to be preserved postoperatively. As a result, these patients benefit from combined electric-acoustic stimulation (EAS) postoperatively. However, 30% to 40% of EAS CI users experience a partial loss of hearing up to 30 dB after surgery. This additional hearing loss is generally not severe enough to preclude use of acoustic amplification; however, it can still impact EAS benefits. The use of electrocochleography (ECoG) measures of peripheral hair cell and neural auditory function have shed insight into the pathophysiology of postimplant loss of residual acoustic hearing. The present study aims to assess the long-term stability of ECoG measures and to establish ECoG as an objective method of monitoring residual hearing over the course of EAS CI use. We hypothesize that repeated measures of ECoG should remain stable over time for EAS CI users with stable postoperative hearing preservation. We also hypothesize that changes in behavioral audiometry for EAS CI users with loss of residual hearing should also be reflected in changes in ECoG measures. DESIGN: A pool of 40 subjects implanted under hearing preservation protocol was included in the study. Subjects were seen at postoperative visits for behavioral audiometry and ECoG recordings. Test sessions occurred 0.5, 1, 3, 6, 12 months, and annually after 12 months postoperatively. Changes in pure-tone behavioral audiometric thresholds relative to baseline were used to classify subjects into two groups: one group with stable acoustic hearing and another group with loss of acoustic hearing. At each test session, ECoG amplitude growth functions for several low-frequency stimuli were obtained. The threshold, slope, and suprathreshold amplitude at a fixed stimulation level was obtained from each growth function at each time point. Longitudinal linear mixed effects models were used to study trends in ECoG thresholds, slopes, and amplitudes for subjects with stable hearing and subjects with hearing loss. RESULTS: Preoperative, behavioral audiometry indicated that subjects had an average low-frequency pure-tone average (125 to 500 Hz) of 40.88 ± 13.12 dB HL. Postoperatively, results showed that ECoG thresholds and amplitudes were stable in EAS CI users with preserved residual hearing. ECoG thresholds increased (worsened) while ECoG amplitudes decreased (worsened) for those with delayed hearing loss. The slope did not distinguish between EAS CI users with stable hearing and subjects with delayed loss of hearing. CONCLUSIONS: These results provide a new application of postoperative ECoG as an objective tool to monitor residual hearing and understand the pathophysiology of delayed hearing loss. While our measures were conducted with custom-designed in-house equipment, CI companies are also designing and implementing hardware and software adaptations to conduct ECoG recordings. Thus, postoperative ECoG recordings can potentially be integrated into clinical practice.

Who's afraid of the X? Incorporating the X and Y chromosomes into the analysis of DNA methylation array data.

BACKGROUND: Many human disease phenotypes manifest differently by sex, making the development of methods for incorporating X and Y-chromosome data into analyses vital. Unfortunately, X and Y chromosome data are frequently excluded from large-scale analyses of the human genome and epigenome due to analytical complexity associated with sex chromosome dosage differences between XX and XY individuals, and the impact of X-chromosome inactivation (XCI) on the epigenome. As such, little attention has been given to considering the methods by which sex chromosome data may be included in analyses of DNA methylation (DNAme) array data. RESULTS: With Illumina Infinium HumanMethylation450 DNAme array data from 634 placental samples, we investigated the effects of probe filtering, normalization, and batch correction on DNAme data from the X and Y chromosomes. Processing steps were evaluated in both mixed-sex and sex-stratified subsets of the analysis cohort to identify whether including both sexes impacted processing results. We found that identification of probes that have a high detection p-value, or that are non-variable, should be performed in sex-stratified data subsets to avoid over- and under-estimation of the quantity of probes eligible for removal, respectively. All normalization techniques investigated returned X and Y DNAme data that were highly correlated with the raw data from the same samples. We found no difference in batch correction results after application to mixed-sex or sex-stratified cohorts. Additionally, we identify two analytical methods suitable for XY chromosome data, the choice between which should be guided by the research question of interest, and we performed a proof-of-concept analysis studying differential DNAme on the X and Y chromosome in the context of placental acute chorioamnionitis. Finally, we provide an annotation of probe types that may be desirable to filter in X and Y chromosome analyses, including probes in repetitive elements, the X-transposed region, and cancer-testis gene promoters. CONCLUSION: While there may be no single "best" approach for analyzing DNAme array data from the X and Y chromosome, analysts must consider key factors during processing and analysis of sex chromosome data to accommodate the underlying biology of these chromosomes, and the technical limitations of DNA methylation arrays.

Derivation of a minimal functional XIST by combining human and mouse interaction domains.

X-inactive specific transcript (XIST) is a 17-19 kb long non-coding ribonucleic acid (RNA) critical for X-chromosome inactivation. Tandem repeats within the RNA serve as functional domains involved in the cis-limited recruitment of heterochromatic changes and silencing. To explore the sufficiency of these domains while generating a functional mini-XIST for targeted silencing approaches, we tested inducible constructs integrated into 8p in a male cell line. Previous results suggested silencing could be accomplished with a transgene comprised of the repeat A, which is highly conserved and critical for silencing; the repeat F that overlaps regulatory elements and the repeat E that contributes to XIST localization by binding proteins such as CIZ1 (AFE). As polycomb-repressive complex 1 (PRC1) is recruited through HNRNPK binding of repeats B-C-D, we included a second 'mini-XIST' comprising AFE with the mouse Polycomb Interaction Domain (PID), a 660-nucleotide region known to recruit PRC1. Silencing of an adjacent gene was possible with and without PID; however, silencing more distally required the addition of PID. The recruitment of heterochromatic marks, evaluated by immunofluorescence combined with RNA fluorescence in situ hybridization, revealed that the AFE domains were sufficient only for CIZ1 recruitment. However, mini-XIST transgene recruited all marks, albeit not to full XIST levels. The ability of the PID domain to facilitate silencing and heterochromatic mark recruitment was unexpected, and inhibition of PRC1 suggested that many of these are PRC1 independent. These results suggest that the addition of this small region allowed the partial recruitment of all the features induced by a full XIST, demonstrating the feasibility of finding a minimal functional XIST.

Acoustic Change Complex Recorded in Hybrid Cochlear Implant Users.

INTRODUCTION: Expanding cochlear implant (CI) candidacy criteria and advances in electrode arrays and soft surgical techniques have increased the number of CI recipients who have residual low-frequency hearing. Objective measures such as obligatory cortical auditory-evoked potentials (CAEPs) may help clinicians make more tailored recommendations to recipients regarding optimal listening mode. As a step toward this goal, this study investigated how CAEPs measured from hybrid CI users differ in two listening modes: acoustic alone (A-alone) versus acoustic plus electric (A + E). METHODS: Eight successful hybrid CI users participated in this study. Two CAEPs, the P1-N1-P2 and the acoustic change complex (ACC), were measured simultaneously in response to the onset and change of a series of different and spectrally complex acoustic signals, in each of the two listening modes (A-alone and A + E). We examined the effects of listening mode and stimulus type on the onset and ACC N1-P2 amplitudes and peak latencies. RESULTS: ACC amplitudes in hybrid CI users significantly differed as a function of listening mode and stimulus type. ACC responses in A + E were larger than those in the A-alone mode. This was most evident for stimuli involving a change from low to high frequency. CONCLUSIONS: Results of this study showed that the ACC varies as a function of listening mode and stimulus type. This finding suggests that the ACC can be used as a physiologic, objective measure of the benefit of hybrid CIs, potentially supporting clinicians in making clinical recommendations on individualized listening mode, or to document subjective preference for a given listening mode. Further research into this potential clinical application in a range of hybrid recipients and/or long electrode users who have residual low-frequency hearing is warranted.

Considering Fish as Recipients of Ecosystem Services Provides a Framework to Formally Link Baseline, Development, and Post-operational Monitoring Programs and Improve Aquatic Impact Assessments for Large Scale Developments.

In most countries, major development projects must satisfy an Environmental Impact Assessment (EIA) process that considers positive and negative aspects to determine if it meets environmental standards and appropriately mitigates or offsets negative impacts on the values being considered. The benefits of before-after-control-impact monitoring designs have been widely known for more than 30 years, but most development assessments fail to effectively link pre- and post-development monitoring in a meaningful way. Fish are a common component of EIA evaluation for both socioeconomic and scientific reasons. The Ecosystem Services (ES) concept was developed to describe the ecosystem attributes that benefit humans, and it offers the opportunity to develop a framework for EIA that is centred around the needs of and benefits from fish. Focusing an environmental monitoring framework on the critical needs of fish could serve to better align risk, development, and monitoring assessment processes. We define the ES that fish provide in the context of two common ES frameworks. To allow for linkages between environmental assessment and the ES concept, we describe critical ecosystem functions from a fish perspective to highlight potential monitoring targets that relate to fish abundance, diversity, health, and habitat. Finally, we suggest how this framing of a monitoring process can be used to better align aquatic monitoring programs across pre-development, development, and post-operational monitoring programs.

Multiple distinct domains of human XIST are required to coordinate gene silencing and subsequent heterochromatin formation.

BACKGROUND: Mammalian dosage compensation is achieved by the inactivation of one X chromosome in XX individuals. In eutheria this process is initiated early in development by the long non-coding RNA XIST. Studies of the initiation of silencing by XIST have focussed on mouse models, so the domains of XIST required to induce silencing in humans, and their relationship with domains required to establish heterochromatin remain to be determined. METHODS: We have previously established an inducible XIST cDNA in somatic cells and shown it can induce silencing and recruit heterochromatic features. We now assess a series of deletions across the transgene for the ability to induce silencing and integrate these results with time-course and chromatin-remodelling inhibitor treatments to follow the steps of XIST-induced silencing and heterochromatinization. DISCUSSION: We find that in addition to the previously reported necessity of the 5' A repeat region for XIST-induced silencing, the 1 kb around the small F repeat region and a non-repetitive region at the 3' end of the RNA are also required to silence genes. Silencing of genes up to 17 Mb from the XIST integration occurs within 2 days, while formation of a Cot-1 depleted domain is slower, and more dependent on the region encompassing Repeat F. The role of this region encompassing Repeat F in both the silencing of actively transcribed genes, the spread of H3K27me3 and the formation of a transcriptionally inert domain suggests a role in a pathway crucial for the spread of XIST across the chromatin to target distal regions of inactivation. Histone deacetylation requires only the A repeat region, with HDAC3 inhibition showing limited effect on silencing, but an impact on H3K27me3 recruitment, and as a result the recruitment of MacroH2A. Global HDAC inhibition impacted silencing in both a distance and dose-dependent fashion. The E repeat region was required for CIZ1 and H4K20me1 recruitment as well as H3K27me3; however, these appeared to act relatively independently. The H3K27me3 mark established by PRC2 integrated silencing and many of the heterochromatic features, while the PRC1 mark ubH2A appeared to be downstream of silencing in these human somatic cells.

Access and Polarization Electrode Impedance Changes in Electric-Acoustic Stimulation Cochlear Implant Users with Delayed Loss of Acoustic Hearing.

Acoustic hearing can be preserved after cochlear implant (CI) surgery, allowing for combined electric-acoustic stimulation (EAS) and superior speech understanding compared to electric-only hearing. Among patients who initially retain useful acoustic hearing, 30-40 % experience a delayed hearing loss that occurs 3 or more months after CI activation. Increases in electrode impedances have been associated with delayed loss of residual acoustic hearing, suggesting a possible role of intracochlear inflammation/fibrosis as reported by Scheperle et al. (Hear Res 350:45-57, 2017) and Shaul et al. (Otol Neurotol 40(5):e518-e526, 2019). These studies measured only total impedance. Total impedance consists of a composite of access resistance, which reflects resistance of the intracochlear environment, and polarization impedance, which reflects resistive and capacitive properties of the electrode-electrolyte interface as described by Dymond (IEEE Trans Biomed Eng 23(4):274-280, 1976) and Tykocinski et al. (Otol Neurotol 26(5):948-956, 2005). To explore the role of access and polarization impedance components in loss of residual acoustic hearing, these measures were collected from Nucleus EAS CI users with stable acoustic hearing and subsequent precipitous loss of hearing. For the hearing loss group, total impedance and access resistance increased over time while polarization impedance remained stable. For the stable hearing group, total impedance and access resistance were stable while polarization impedance declined. Increased access resistance rather than polarization impedance appears to drive the increase in total impedances seen with loss of hearing. Moreover, access resistance has been correlated with intracochlear fibrosis/inflammation in animal studies as observed by Xu et al. (Hear Res 105(1-2):1-29, 1997) and Tykocinski et al. (Hear Res 159(1-2):53-68, 2001). These findings thus support intracochlear inflammation as one contributor to loss of acoustic hearing in our EAS CI population.

Pterostilbene leads to DNMT3B-mediated DNA methylation and silencing of OCT1-targeted oncogenes in breast cancer cells.

Transcription factor (TF)-mediated regulation of genes is often disrupted during carcinogenesis. The DNA methylation state of TF-binding sites may dictate transcriptional activity of corresponding genes. Stilbenoid polyphenols, such as pterostilbene (PTS), have been shown to exert anticancer action by remodeling DNA methylation and gene expression. However, the mechanisms behind these effects still remain unclear. Here, the dynamics between oncogenic TF OCT1 binding and de novo DNA methyltransferase DNMT3B binding in PTS-treated MCF10CA1a invasive breast cancer cells has been explored. Using chromatin immunoprecipitation (ChIP) followed by next generation sequencing, we determined 47 gene regulatory regions with decreased OCT1 binding and enriched DNMT3B binding in response to PTS. Most of those genes were found to have oncogenic functions. We selected three candidates, PRKCA, TNNT2, and DANT2, for further mechanistic investigation taking into account PRKCA functional and regulatory connection with numerous cancer-driving processes and pathways, and some of the highest increase in DNMT3B occupancy within TNNT2 and DANT2 enhancers. PTS led to DNMT3B recruitment within PRKCA, TNNT2, and DANT2 at loci that also displayed reduced OCT1 binding. Substantial decrease in OCT1 with increased DNMT3B binding was accompanied by PRKCA promoter and TNNT2 and DANT2 enhancer hypermethylation, and gene silencing. Interestingly, DNA hypermethylation of the genes was not detected in response to PTS in DNMT3B-CRISPR knockout MCF10CA1a breast cancer cells. It indicates DNMT3B-dependent methylation of PRKCA, TNNT2, and DANT2 upon PTS. Our findings provide a better understanding of mechanistic players and their gene targets that possibly contribute to the anticancer action of stilbenoid polyphenols.

Contribution of genetic and epigenetic changes to escape from X-chromosome inactivation.

BACKGROUND: X-chromosome inactivation (XCI) is the epigenetic inactivation of one of two X chromosomes in XX eutherian mammals. The inactive X chromosome is the result of multiple silencing pathways that act in concert to deposit chromatin changes, including DNA methylation and histone modifications. Yet over 15% of genes escape or variably escape from inactivation and continue to be expressed from the otherwise inactive X chromosome. To the extent that they have been studied, epigenetic marks correlate with this expression. RESULTS: Using publicly available data, we compared XCI status calls with DNA methylation, H3K4me1, H3K4me3, H3K9me3, H3K27ac, H3K27me3 and H3K36me3. At genes subject to XCI we found heterochromatic marks enriched, and euchromatic marks depleted on the inactive X when compared to the active X. Genes escaping XCI were more similar between the active and inactive X. Using sample-specific XCI status calls, we found some marks differed significantly with variable XCI status, but which marks were significant was not consistent between genes. A model trained to predict XCI status from these epigenetic marks obtained over 75% accuracy for genes escaping and over 90% for genes subject to XCI. This model made novel XCI status calls for genes without allelic differences or CpG islands required for other methods. Examining these calls across a domain of variably escaping genes, we saw XCI status vary across individual genes rather than at the domain level. Lastly, we compared XCI status calls to genetic polymorphisms, finding multiple loci associated with XCI status changes at variably escaping genes, but none individually sufficient to induce an XCI status change. CONCLUSION: The control of expression from the inactive X chromosome is multifaceted, but ultimately regulated at the individual gene level with detectable but limited impact of distant polymorphisms. On the inactive X, at silenced genes euchromatic marks are depleted while heterochromatic marks are enriched. Genes escaping inactivation show a less significant enrichment of heterochromatic marks and depletion of H3K27ac. Combining all examined marks improved XCI status prediction, particularly for genes without CpG islands or polymorphisms, as no single feature is a consistent feature of silenced or expressed genes.

A cross-cohort analysis of autosomal DNA methylation sex differences in the term placenta.

BACKGROUND: Human placental DNA methylation (DNAme) data is a valuable resource for studying sex differences during gestation, as DNAme profiles after delivery reflect the cumulative effects of gene expression patterns and exposures across gestation. Here, we present an analysis of sex differences in autosomal DNAme in the uncomplicated term placenta (n = 343) using the Illumina 450K array. RESULTS: At a false discovery rate < 0.05 and a mean sex difference in DNAme beta value of > 0.10, we identified 162 autosomal CpG sites that were differentially methylated by sex and replicated in an independent cohort of samples (n = 293). Several of these differentially methylated CpG sites were part of larger correlated regions of sex differential DNAme. Although global DNAme levels did not differ by sex, the majority of significantly differentially methylated CpGs were more highly methylated in male placentae, the opposite of what is seen in differential methylation analyses of somatic tissues. Patterns of autosomal DNAme at these 162 CpGs were significantly associated with maternal age (in males) and newborn birthweight standard deviation (in females). CONCLUSIONS: Our results provide a comprehensive analysis of sex differences in autosomal DNAme in the term human placenta. We report a list of high-confidence autosomal sex-associated differentially methylated CpGs and identify several key features of these loci that suggest their relevance to sex differences observed in normative and complicated pregnancies.

Independent domains for recruitment of PRC1 and PRC2 by human XIST.

XIST establishes inactivation across its chromosome of origin, even when expressed from autosomal transgenes. To identify the regions of human XIST essential for recruiting heterochromatic marks we generated a series of overlapping deletions in an autosomal inducible XIST transgene present in 8p of the HT1080 male fibrosarcoma cell line. We examined the ability of each construct to enrich its unified XIST territory with the histone marks established by PRC1 and PRC2 as well as the heterochromatin factors MacroH2A and SMCHD1. Chromatin enrichment of ubH2A by PRC1 required four distinct regions of XIST, and these were completely distinct from the two domains crucial for enrichment of H3K27me3 by PRC2. Both the domains required, as well as the impact of PRC1 and PRC2 inhibitors, suggest that PRC1 is required for SMCHD1 while PRC2 function is necessary for MacroH2A recruitment, although incomplete overlap of regions implicates roles for additional factors. This cooperativity between factors contributes to the requirement for multiple separate domains being required for each feature examined. The independence of the PRC1/PRC2 pathways was observed when XIST was expressed both autosomally or from the X chromosome suggesting that these observations are not purely a result of the context in which XIST operates. Although independent domains were required for the PRC1 and PRC2 pathways overall all regions tested were important for some aspect of XIST functionality, demonstrating both modularity and cooperativity across the XIST lncRNA.

Residual Hair Cell Responses in Electric-Acoustic Stimulation Cochlear Implant Users with Complete Loss of Acoustic Hearing After Implantation.

Changes in cochlear implant (CI) design and surgical techniques have enabled the preservation of residual acoustic hearing in the implanted ear. While most Nucleus Hybrid L24 CI users retain significant acoustic hearing years after surgery, 6-17 % experience a complete loss of acoustic hearing (Roland et al. Laryngoscope. 126(1):175-81. (2016), Laryngoscope. 128(8):1939-1945 (2018); Scheperle et al. Hear Res. 350:45-57 (2017)). Electrocochleography (ECoG) enables non-invasive monitoring of peripheral auditory function and may provide insight into the pathophysiology of hearing loss. The ECoG response is evoked using an acoustic stimulus and includes contributions from the hair cells (cochlear microphonic-CM) as well as the auditory nerve (auditory nerve neurophonic-ANN). Seven Hybrid L24 CI users with complete loss of residual hearing months after surgery underwent ECoG measures before and after loss of hearing. While significant reductions in CMs were evident after hearing loss, all participants had measurable CMs despite having no measurable acoustic hearing. None retained measurable ANNs. Given histological data suggesting stable hair cell and neural counts after hearing loss (e.g., Quesnel et al. Hear Res. 333:225-234. (2016)), the loss of ECoG and audiometric hearing may reflect reduced synaptic input. This is consistent with the theory that residual CM responses coupled with little to no ANN responses reflect a "disconnect" between hair cells and auditory nerve fibers (Fontenot et al. Ear Hear. 40(3):577-591. 2019). This "disconnection" may prevent proper encoding of auditory stimulation at higher auditory pathways, leading to a lack of audiometric responses, even in the presence of viable cochlear hair cells.

Cross-species examination of X-chromosome inactivation highlights domains of escape from silencing.

BACKGROUND: X-chromosome inactivation (XCI) in eutherian mammals is the epigenetic inactivation of one of the two X chromosomes in XX females in order to compensate for dosage differences with XY males. Not all genes are inactivated, and the proportion escaping from inactivation varies between human and mouse (the two species that have been extensively studied). RESULTS: We used DNA methylation to predict the XCI status of X-linked genes with CpG islands across 12 different species: human, chimp, bonobo, gorilla, orangutan, mouse, cow, sheep, goat, pig, horse and dog. We determined the XCI status of 342 CpG islands on average per species, with most species having 80-90% of genes subject to XCI. Mouse was an outlier, with a higher proportion of genes subject to XCI than found in other species. Sixteen genes were found to have discordant X-chromosome inactivation statuses across multiple species, with five of these showing primate-specific escape from XCI. These discordant genes tended to cluster together within the X chromosome, along with genes with similar patterns of escape from XCI. CTCF-binding, ATAC-seq signal and LTR repeats were enriched at genes escaping XCI when compared to genes subject to XCI; however, enrichment was only observed in three or four of the species tested. LINE and DNA repeats showed enrichment around subject genes, but again not in a consistent subset of species. CONCLUSIONS: In this study, we determined XCI status across 12 species, showing mouse to be an outlier with few genes that escape inactivation. Inactivation status is largely conserved across species. The clustering of genes that change XCI status across species implicates a domain-level control. In contrast, the relatively consistent, but not universal correlation of inactivation status with enrichment of repetitive elements or CTCF binding at promoters demonstrates gene-based influences on inactivation state. This study broadens enrichment analysis of regulatory elements to species beyond human and mouse.

Comparisons of the Sensitivity and Reliability of Multiple Measures of Listening Effort.

OBJECTIVES: The objective of this study was to evaluate the sensitivity and reliability of one subjective (rating scale) and three objective (dual-task paradigm, pupillometry, and skin conductance response amplitude) measures of listening effort across multiple signal to noise ratios (SNRs). DESIGN: Twenty adults with normal hearing attended two sessions and listened to sentences presented in quiet and in stationary noise at three different SNRs: 0, -3, and -5 dB. Listening effort was assessed by examining change in reaction time (dual-task paradigm), change in peak to peak pupil diameter (pupillometry), and change in mean skin conductance response amplitude; self-reported listening effort on a scale from 0 to 100 was also evaluated. Responses were averaged within each SNR and based on three word recognition ability categories (≤50%, 51% to 71%, and >71%) across all SNRs. Measures were considered reliable if there were no significant changes between sessions, and intraclass correlation coefficients were a minimum of 0.40. Effect sizes were calculated to compare the sensitivity of the measures. RESULTS: Intraclass correlation coefficient values indicated fair-to-moderate reliability for all measures while individual measurement sensitivity was variable. Self-reports were sensitive to listening effort but were less reliable, given that subjective effort was greater during the dual task than either of the physiologic measures. The dual task was sensitive to a narrow range of word recognition abilities but was less reliable as it exhibited a global decrease in reaction time across sessions. Pupillometry was consistently sensitive and reliable to changes in listening effort. Skin conductance response amplitude was not sensitive or reliable while the participants listened to the sentences. Skin conductance response amplitude during the verbal response was sensitive to poor (≤50%) speech recognition abilities; however, it was less reliable as there was a significant change in amplitude across sessions. CONCLUSIONS: In this study, pupillometry was the most sensitive and reliable objective measure of listening effort. Intersession variability significantly influenced the other objective measures of listening effort, which suggests challenges for cross-study comparability. Therefore, intraclass correlation coefficients combined with other statistical tests more fully describe the reliability of measures of listening effort across multiple difficulties. Minimizing intersession variability will increase measurement sensitivity. Further work toward standardized methods and analysis will strengthen our understanding of the reliability and sensitivity of measures of listening effort and better facilitate cross-modal and cross-study comparisons.

Assessment of long non-coding RNA expression reveals novel mediators of the lung tumour immune response.

The tumour immune microenvironment is a crucial mediator of lung tumourigenesis, and characterizing the immune landscape of patient tumours may guide immunotherapy treatment regimens and uncover novel intervention points. We sought to identify the landscape of tumour-infiltrating immune cells in the context of long non-coding RNA (lncRNAs), known regulators of gene expression. We examined the lncRNA profiles of lung adenocarcinoma (LUAD) tumours by interrogating RNA sequencing data from microdissected and non-microdissected samples (BCCRC and TCGA). Subsequently, analysis of single-cell RNA sequencing data from lung tumours and flow-sorted healthy peripheral blood mononuclear cells identified lncRNAs in immune cells, highlighting their biological and prognostic relevance. We discovered lncRNA expression patterns indicative of regulatory relationships with immune-related protein-coding genes, including the relationship between AC008750.1 and NKG7 in NK cells. Activation of NK cells in vitro was sufficient to induce AC008750.1 expression. Finally, siRNA-mediated knockdown of AC008750.1 significantly impaired both the expression of NKG7 and the anti-tumour capacity of NK cells. We present an atlas of cancer-cell extrinsic immune cell-expressed lncRNAs, in vitro evidence for a functional role of lncRNAs in anti-tumour immune activity, which upon further exploration may reveal novel clinical utility as markers of immune infiltration.

Publisher Correction: A planet within the debris disk around the pre-main-sequence star AU Microscopii.

An amendment to this paper has been published and can be accessed via a link at the top of the paper.