Long-Term Adherence to Continuous Positive Airway Pressure in Patients with Obstructive Sleep Apnoea Set Up in a Complete Remote Pathway: A Single-Centre Service Evaluation Project.

Background: Continuous positive airway pressure (CPAP) is the first-line treatment for obstructive sleep apnoea (OSA). Maintaining adherence to CPAP in the long term is a clinical problem, and numerous factors have been identified that impact adherence. Although fully remote diagnostic and CPAP services were frequently utilised during the COVID-19 pandemic for patients with OSA, long-term adherence data have not been published. The aim of this service evaluation project was to describe the long-term adherence to CPAP. We also analysed factors that are associated with it. Methods: two-hundred and eighty patients diagnosed with OSA and set up on CPAP remotely during the first wave of the COVID-19 pandemic as part of routine clinical practice were analysed. Results: One-hundred and seven patients (38%) were fully adherent to CPAP at 24 months, determined by at least 4 h of usage on at least 70% of the days. Of the factors analysed, body mass index, disease severity, driving status and the presence of depression were related to long-term adherence (all p < 0.05). Conclusions: with the likelihood of future pandemics similar to COVID-19, our data provide evidence that fully remote pathways for management of patients with OSA can be designed and be sustainable with good long-term adherence.

The relationship between the price and demand of alcohol, tobacco, unhealthy food, sugar-sweetened beverages, and gambling: an umbrella review of systematic reviews.

BACKGROUND: The WHO highlight alcohol, tobacco, unhealthy food, and sugar-sweetened beverage (SSB) taxes as one of the most effective policies for preventing and reducing the burden of non-communicable diseases. This umbrella review aimed to identify and summarise evidence from systematic reviews that report the relationship between price and demand or price and disease/death for alcohol, tobacco, unhealthy food, and SSBs. Given the recent recognition as gambling as a public health problem, we also included gambling. METHODS: The protocol for this umbrella review was pre-registered (PROSPERO CRD42023447429). Seven electronic databases were searched between 2000-2023. Eligible systematic reviews were those published in any country, including adults or children, and which quantitatively examined the relationship between alcohol, tobacco, gambling, unhealthy food, or SSB price/tax and demand (sales/consumption) or disease/death. Two researchers undertook screening, eligibility, data extraction, and risk of bias assessment using the ROBIS tool. RESULTS: We identified 50 reviews from 5,185 records, of which 31 reported on unhealthy food or SSBs, nine reported on tobacco, nine on alcohol, and one on multiple outcomes (alcohol, tobacco, unhealthy food, and SSBs). We did not identify any reviews on gambling. Higher prices were consistently associated with lower demand, notwithstanding variation in the size of effect across commodities or populations. Reductions in demand were large enough to be considered meaningful for policy. CONCLUSIONS: Increases in the price of alcohol, tobacco, unhealthy food, and SSBs are consistently associated with decreases in demand. Moreover, increasing taxes can be expected to increase tax revenue. There may be potential in joining up approaches to taxation across the harm-causing commodities.

New approach methodologies to enhance human health risk assessment of immunotoxic properties of chemicals - a PARC (Partnership for the Assessment of Risk from Chemicals) project.

As a complex system governing and interconnecting numerous functions within the human body, the immune system is unsurprisingly susceptible to the impact of toxic chemicals. Toxicants can influence the immune system through a multitude of mechanisms, resulting in immunosuppression, hypersensitivity, increased risk of autoimmune diseases and cancer development. At present, the regulatory assessment of the immunotoxicity of chemicals relies heavily on rodent models and a limited number of Organisation for Economic Co-operation and Development (OECD) test guidelines, which only capture a fraction of potential toxic properties. Due to this limitation, various authorities, including the World Health Organization and the European Food Safety Authority have highlighted the need for the development of novel approaches without the use of animals for immunotoxicity testing of chemicals. In this paper, we present a concise overview of ongoing efforts dedicated to developing and standardizing methodologies for a comprehensive characterization of the immunotoxic effects of chemicals, which are performed under the EU-funded Partnership for the Assessment of Risk from Chemicals (PARC).

Australasian Malignant PLeural Effusion (AMPLE)-4 trial: study protocol for a multi-centre randomised trial of topical antibiotics prophylaxis for infections of indwelling pleural catheters.

BACKGROUND: Malignant pleural effusion (MPE) is a debilitating condition as it commonly causes disabling breathlessness and impairs quality of life (QoL). Indwelling pleural catheter (IPC) offers an effective alternative for the management of MPE. However, IPC-related infections remain a significant concern and there are currently no long-term strategies for their prevention. The Australasian Malignant PLeural Effusion (AMPLE)-4 trial is a multicentre randomised trial that evaluates the use of topical mupirocin prophylaxis (vs no mupirocin) to reduce catheter-related infections in patients with MPE treated with an IPC. METHODS: A pragmatic, multi-centre, open-labelled, randomised trial. Eligible patients with MPE and an IPC will be randomised 1:1 to either regular topical mupirocin prophylaxis or no mupirocin (standard care). For the interventional arm, topical mupirocin will be applied around the IPC exit-site after each drainage, at least twice weekly. Weekly follow-up via phone calls or in person will be conducted for up to 6 months. The primary outcome is the percentage of patients who develop an IPC-related (pleural, skin, or tract) infection between the time of catheter insertion and end of follow-up period. Secondary outcomes include analyses of infection (types and episodes), hospitalisation days, health economics, adverse events, and survival. Subject to interim analyses, the trial will recruit up to 418 participants. DISCUSSION: Results from this trial will determine the efficacy of mupirocin prophylaxis in patients who require IPC for MPE. It will provide data on infection rates, microbiology, and potentially infection pathways associated with IPC-related infections. ETHICS AND DISSEMINATION: Sir Charles Gairdner and Osborne Park Health Care Group Human Research Ethics Committee has approved the study (RGS0000005920). Results will be published in peer-reviewed journals and presented at scientific conferences. TRIAL REGISTRATION: Australia New Zealand Clinical Trial Registry ACTRN12623000253606. Registered on 9 March 2023.

Predicting the intention to receive the COVID-19 booster vaccine based on the health belief model.

COVID-19 vaccine boosters are recommended because the protection provided by previous doses eventually decreases, posing a threat to immunity. Some people, however, remain hesitant or unwilling to get vaccinated. The present study sought to investigate factors associated with the intention to receive the COVID-19 booster vaccine based on (1) the constructs of the Health Belief Model, and (2) trust in healthcare workers and science. A sample of 165 adults with two doses of the COVID-19 vaccine were recruited using convenience sampling. Data was collected using an online survey from November 2021 to January 2022. The survey included questions about participants' socio-demographic details, health beliefs, trust, history of COVID-19 vaccination, and the intention to receive a third dose of the COVID-19 vaccine. Ordinal logistic regression analysis showed that higher perceived benefits, severity, and trust in healthcare workers, and lower perceived barriers predicted higher willingness to get a third dose of the vaccine whereas perceived susceptibility and trust in science did not. Understanding the factors and health beliefs that underlie vaccine hesitancy are vital when developing effective interventions with the aim of increasing uptake of COVID-19 booster vaccines.

Comprehensive mapping of the AOP-Wiki database: identifying biological and disease gaps.

Introduction: The Adverse Outcome Pathway (AOP) concept facilitates rapid hazard assessment for human health risks. AOPs are constantly evolving, their number is growing, and they are referenced in the AOP-Wiki database, which is supported by the OECD. Here, we present a study that aims at identifying well-defined biological areas, as well as gaps within the AOP-Wiki for future research needs. It does not intend to provide a systematic and comprehensive summary of the available literature on AOPs but summarizes and maps biological knowledge and diseases represented by the already developed AOPs (with OECD endorsed status or under validation). Methods: Knowledge from the AOP-Wiki database were extracted and prepared for analysis using a multi-step procedure. An automatic mapping of the existing information on AOPs (i.e., genes/proteins and diseases) was performed using bioinformatics tools (i.e., overrepresentation analysis using Gene Ontology and DisGeNET), allowing both the classification of AOPs and the development of AOP networks (AOPN). Results: AOPs related to diseases of the genitourinary system, neoplasms and developmental anomalies are the most frequently investigated on the AOP-Wiki. An evaluation of the three priority cases (i.e., immunotoxicity and non-genotoxic carcinogenesis, endocrine and metabolic disruption, and developmental and adult neurotoxicity) of the EU-funded PARC project (Partnership for the Risk Assessment of Chemicals) are presented. These were used to highlight under- and over-represented adverse outcomes and to identify and prioritize gaps for further research. Discussion: These results contribute to a more comprehensive understanding of the adverse effects associated with the molecular events in AOPs, and aid in refining risk assessment for stressors and mitigation strategies. Moreover, the FAIRness (i.e., data which meets principles of findability, accessibility, interoperability, and reusability (FAIR)) of the AOPs appears to be an important consideration for further development.

Differentiation signals induce APOBEC3A expression via GRHL3 in squamous epithelia and squamous cell carcinoma.

Two APOBEC (apolipoprotein-B mRNA editing enzyme catalytic polypeptide-like) DNA cytosine deaminase enzymes (APOBEC3A and APOBEC3B) generate somatic mutations in cancer, driving tumour development and drug resistance. Here we used single cell RNA sequencing to study APOBEC3A and APOBEC3B expression in healthy and malignant mucosal epithelia, validating key observations with immunohistochemistry, spatial transcriptomics and functional experiments. Whereas APOBEC3B is expressed in keratinocytes entering mitosis, we show that APOBEC3A expression is confined largely to terminally differentiating cells and requires Grainyhead-like transcription factor 3 (GRHL3). Thus, in normal tissue, neither deaminase appears to be expressed at high levels during DNA replication, the cell cycle stage associated with APOBEC-mediated mutagenesis. In contrast, we show that in squamous cell carcinoma tissues, there is expansion of GRHL3 expression and activity to a subset of cells undergoing DNA replication and concomitant extension of APOBEC3A expression to proliferating cells. These findings indicate a mechanism for acquisition of APOBEC3A mutagenic activity in tumours.

Facilitating knowledge transfer to policy makers and front-line workers during a pandemic: Implementation, impact and lessons learned.

BACKGROUND: Stakeholders working on the COVID-19 pandemic response needed access to evidence, requiring a systematic approach to identify and disseminate relevant research. OBJECTIVES: Outline the stages of development of a COVID-19 Literature Digest; demonstrate the impact the Digest had on decision-making and knowledge gain; identify the lessons learned. METHODS: A standardised process was developed to identify and select papers. The main sources for content were PubMed, bioRxiv and medRxiv. A shared EndNote library was used to deduplicate and organise papers. Three user surveys obtained feedback from subscribers to determine if the Digest remained valuable, and explore the benefits to individuals. RESULTS: 40-60 papers were summarised each week. 211 Digests were produced from March 2020 to March 2022, with around 10,000 papers included altogether. Survey results suggest benefits of the Digest were gaining new knowledge, saving time and contributing to evidence-based decision making. DISCUSSION: Digest procedures constantly evolved and were adapted in response to survey feedback. Lessons identified: learn from failure, communication is key, measure your impact, work collaboratively, reflect and be flexible. CONCLUSION: The Digest was successfully produced within the limits of available resource. The learning from this Digest will inform evidence monitoring, selection and dissemination for future health crises.

Assessment of global antimicrobial resistance campaigns conducted to improve public awareness and antimicrobial use behaviours: a rapid systematic review.

INTRODUCTION: Public health campaigns with a well-defined outcome behaviour have been shown to successfully alter behaviour. However, the complex nature of antimicrobial resistance (AMR) creates challenges when evaluating campaigns aimed at raising awareness and changing behaviour. AIMS: To determine what campaigns have been conducted and which reported being effective at improving awareness of antimicrobial resistance and changing behaviour around antimicrobial use in members of the public. It also sought to determine the outcome measures studies have used to assess campaign effectiveness. METHODS: A systematic search of Ovid MEDLINE and Embase, was conducted in October 2022 using a predefined search strategy. Studies which were published between 2010 and September 2022 that outlined a campaign or invention aimed at the public and focusing on AMR or antibiotic usage were eligible for inclusion and studies which solely targeted healthcare professionals (HCP) were excluded. RESULTS: Literature searches retrieved 6961 results. De-duplication and screening removed 6925 articles, five articles from grey literature and reference screening were included, giving a total of 41 studies and 30 unique interventions. There was a distribution of campaigns globally with the majority run in Europe (n = 15) with most campaigns were conducted nationally (n = 14). Campaigns tended to focus on adult members of the public (n = 14) or targeted resources towards both the public and HCPs (n = 13) and predominately assessed changes in knowledge of and/or attitudes towards AMR (n = 16). Campaigns where an improvement was seen in their primary outcome measure tended to use mass media to disseminate information, targeted messaging towards a specific infection, and including the use of HCP-patient interactions. DISCUSSION: This review provides some evidence that campaigns can significantly improve outcome measures relating to AMR and antibiotic usage. Despite a lack of homogeneity between studies some common themes emerged between campaigns reported as being effective. However, the frequent use of observational study designs makes it difficult to establish causation between the campaign and changes seen in the studies outcome measures. It is important that clear evaluation processes are embedded as part of the design process for future campaigns; a campaign evaluation framework for use by campaign developers may facilitate this.

Beyond pGALS: the need for a multifaceted musculoskeletal decision-making tool ('pGALSplus') in community-based clinical practice.

Musculoskeletal (MSK) problems in children are common, and health-care professionals must identify those requiring onward referral. Paediatric gait, arms, legs and spine (pGALS) is an MSK assessment to discern abnormal joints. We aimed to identify MSK assessments to add to pGALS (pGALSplus) to facilitate decision-making in the context of exemplar conditions representing a spectrum of MSK presentations, namely JIA, mucopolysaccharidoses, muscular dystrophy and developmental co-ordination disorder. A literature review identified 35 relevant articles that focused on clinical assessments [including questionnaire(s), physical examination and functional tests] used by health-care professionals in the context of the exemplar conditions. We provide a description of these assessments and the rationale regarding how they, or components of such tools, might be useful within pGALSplus. This process provides a foundation for further work to develop and validate pGALSplus.

Exploring informal caregivers' well-being during COVID-19 through online discussion forums.

BACKGROUND: COVID-19 has increased pressures on caregivers, disruptions to health services and increased health concerns during COVID-19. Reports have been made on informal carers' increased workload and limited support services during the pandemic. AIMS: This study aimed to explore how informal caregivers experienced their well-being during COVID-19 through online discussion forums. MATERIALS AND METHODS: A reflexive thematic analysis characterised by theoretical flexibility, organic inductive coding processes and theme development was conducted on online discussion forums. The method highlighted theme reviewing which was done twice to encourage data reflection. The project was conducted on a novel topic which was a new area of research interest. Semantic coding where participants' words were used directly in the interpretation and construction of themes was used. RESULTS: In the theme 'Locked in or locked away' caregivers worried about continuing care at home, due to limited freedom and worries of hiring help during a pandemic. Some expressed worries about visitation rights and grief of not being present with a loved one if they would reside in a care home. The theme 'Nothing left to give' suggested that COVID-19 exasperated caregivers' loneliness, social isolation and increased responsibilities and challenges with other roles. Bitterness, resentment and anger were felt towards lack of social support and workload. Theme 'Celebrating a virtual way of life' described how caregivers used online forums when other support services were disrupted. DISCUSSION: We discuss the role of informal caregiver that was described as all-encompassing during COVID-19. We highlight the importance of advanced planning for care home transitions and the use of online forums as a form of support. We suggest further exploration into informal caregivers' role balancing. CONCLUSION: COVID-19 seemed to affect informal caregivers negatively, but they reframed their situations and sought online support. With COVID-19-related restrictions and increased workload, COVID-19 added an all-or-nothing aspect to care home transition decisions.

Hazard characterization of Alternaria toxins to identify data gaps and improve risk assessment for human health.

Fungi of the genus Alternaria are ubiquitous plant pathogens and saprophytes which are able to grow under varying temperature and moisture conditions as well as on a large range of substrates. A spectrum of structurally diverse secondary metabolites with toxic potential has been identified, but occurrence and relative proportion of the different metabolites in complex mixtures depend on strain, substrate, and growth conditions. This review compiles the available knowledge on hazard identification and characterization of Alternaria toxins. Alternariol (AOH), its monomethylether AME and the perylene quinones altertoxin I (ATX-I), ATX-II, ATX-III, alterperylenol (ALP), and stemphyltoxin III (STTX-III) showed in vitro genotoxic and mutagenic properties. Of all identified Alternaria toxins, the epoxide-bearing analogs ATX-II, ATX-III, and STTX-III show the highest cytotoxic, genotoxic, and mutagenic potential in vitro. Under hormone-sensitive conditions, AOH and AME act as moderate xenoestrogens, but in silico modeling predicts further Alternaria toxins as potential estrogenic factors. Recent studies indicate also an immunosuppressive role of AOH and ATX-II; however, no data are available for the majority of Alternaria toxins. Overall, hazard characterization of Alternaria toxins focused, so far, primarily on the commercially available dibenzo-α-pyrones AOH and AME and tenuazonic acid (TeA). Limited data sets are available for altersetin (ALS), altenuene (ALT), and tentoxin (TEN). The occurrence and toxicological relevance of perylene quinone-based Alternaria toxins still remain to be fully elucidated. We identified data gaps on hazard identification and characterization crucial to improve risk assessment of Alternaria mycotoxins for consumers and occupationally exposed workers.

Structural basis of promiscuous substrate transport by Organic Cation Transporter 1.

Organic Cation Transporter 1 (OCT1) plays a crucial role in hepatic metabolism by mediating the uptake of a range of metabolites and drugs. Genetic variations can alter the efficacy and safety of compounds transported by OCT1, such as those used for cardiovascular, oncological, and psychological indications. Despite its importance in drug pharmacokinetics, the substrate selectivity and underlying structural mechanisms of OCT1 remain poorly understood. Here, we present cryo-EM structures of full-length human OCT1 in the inward-open conformation, both ligand-free and drug-bound, indicating the basis for its broad substrate recognition. Comparison of our structures with those of outward-open OCTs provides molecular insight into the alternating access mechanism of OCTs. We observe that hydrophobic gates stabilize the inward-facing conformation, whereas charge neutralization in the binding pocket facilitates the release of cationic substrates. These findings provide a framework for understanding the structural basis of the promiscuity of drug binding and substrate translocation in OCT1.

Gene expression analyses of TAS1R taste receptors relevant to the treatment of cardiometabolic disease.

The sweet taste receptor (STR) is a G protein-coupled receptor (GPCR) responsible for mediating cellular responses to sweet stimuli. Early evidence suggests that elements of the STR signaling system are present beyond the tongue in metabolically active tissues, where it may act as an extraoral glucose sensor. This study aimed to delineate expression of the STR in extraoral tissues using publicly available RNA-sequencing repositories. Gene expression data was mined for all genes implicated in the structure and function of the STR, and control genes including highly expressed metabolic genes in relevant tissues, other GPCRs and effector G proteins with physiological roles in metabolism, and other GPCRs with expression exclusively outside the metabolic tissues. Since the physiological role of the STR in extraoral tissues is likely related to glucose sensing, expression was then examined in diseases related to glucose-sensing impairment such as type 2 diabetes. An aggregate co-expression network was then generated to precisely determine co-expression patterns among the STR genes in these tissues. We found that STR gene expression was negligible in human pancreatic and adipose tissues, and low in intestinal tissue. Genes encoding the STR did not show significant co-expression or connectivity with other functional genes in these tissues. In addition, STR expression was higher in mouse pancreatic and adipose tissues, and equivalent to human in intestinal tissue. Our results suggest that STR expression in mice is not representative of expression in humans, and the receptor is unlikely to be a promising extraoral target in human cardiometabolic disease.

Staphylococcus capitis: Review of Its Role in Infections and Outbreaks.

In June 2021, a national incident team was formed due to an increased detection of Staphylococcus capitis in samples from hospitalised infants. Staphylococcus capitis has been known to cause outbreaks in neonatal units across the globe, but the extent of the UK spread was unclear. A literature review was undertaken to support case identification, clinical management and environmental infection control. A literature search was undertaken on multiple databases from inception to 24 May 2021, using keywords such as "Staphylococcus capitis", "NRCS-A", "S. capitis", "neonate", "newborn" and "neonatal intensive care unit" (NICU). After screening, 223 articles of relevance were included. Results show incidences of S. capitis outbreaks have frequently been associated with the outbreak clone (NRCS-A) and environmental sources. The NRCS-A harbours a multidrug resistance profile that includes resistance to beta-lactam antibiotics and aminoglycosides, with several papers noting resistance or heteroresistance to vancomycin. The NRCS-A clone also harbours a novel SCCmec-SCCcad/ars/cop composite island and increased vancomycin resistance. The S. capitis NRCS-A clone has been detected for decades, but the reasons for the potentially increased frequency are unclear, as are the most effective interventions to manage outbreaks associated with this clone. This supports the need for improvements in environmental control and decontamination strategies to prevent transmission.

Critical appraisal in rapid systematic reviews of COVID-19 studies: implementation of the Quality Criteria Checklist (QCC).

In this letter, we briefly describe how we selected and implemented the quality criteria checklist (QCC) as a critical appraisal tool in rapid systematic reviews conducted to inform public health advice, guidance and policy during the COVID-19 pandemic. As these rapid reviews usually included a range of study designs, it was key to identify a single tool that would allow for reliable critical appraisal across most experimental and observational study designs and applicable to a range of topics. After carefully considering a number of existing tools, the QCC was selected as it had good interrater agreement between three reviewers (Fleiss kappa coefficient 0.639) and was found to be easy and fast to apply once familiar with the tool. The QCC consists of 10 questions, with sub-questions to specify how it should be applied to a specific study design. Four of these questions are considered as critical (on selection bias, group comparability, intervention/exposure assessment and outcome assessment) and the rating of a study (high, moderate or low methodological quality) depends on the responses to these four critical questions. Our results suggest that the QCC is an appropriate critical appraisal tool to assess experimental and observational studies within COVID-19 rapid reviews. This study was done at pace during the COVID-19 pandemic; further reliability analyses should be conducted, and more research is needed to validate the QCC across a range of public health topics.

Primary cilia, A-kinase anchoring proteins and constitutive activity at the orphan G protein-coupled receptor GPR161: A tale about a tail.

Primary cilia are non-motile antennae-like structures responsible for sensing environmental changes in most mammalian cells. Ciliary signalling is largely mediated by the Sonic Hedgehog (Shh) pathway, which acts as a master regulator of ciliary protein transit and is essential for normal embryonic development. One particularly important player in primary cilia is the orphan G protein-coupled receptor, GPR161. In this review, we introduce GPR161 in the context of Shh signalling and describe the unique features on its C-terminus such as PKA phosphorylation sites and an A-kinase anchoring protein motif, which may influence the function of the receptor, cAMP compartmentalisation and/or trafficking within primary cilia. We discuss the recent putative pairing of GPR161 and spexin-1, highlighting the additional steps needed before GPR161 could be considered 'deorphanised'. Finally, we speculate that the marked constitutive activity and unconventional regulation of GPR161 may indicate that the receptor may not require an endogenous ligand.

Type 2 diabetes influences intraepithelial corneal nerve parameters and corneal stromal-epithelial nerve penetration sites.

INTRODUCTION: The quantification of intraepithelial corneal basal nerve parameters by in vivo confocal microscopy represents a promising modality to identify the earliest manifestations of diabetic peripheral neuropathy. However, its diagnostic accuracy is hampered by its dependence on neuron length, with minimal consideration for other parameters, including the origin of these nerves, the corneal stromal-epithelial nerve penetration sites. This study sought to utilize high-resolution images of murine corneal nerves to analyze comprehensively the morphological changes associated with type 2 diabetes progression. MATERIALS AND METHODS: ßIII-Tubulin immunostained corneas from prediabetic and type 2 diabetic mice and their respective controls were imaged by scanning confocal microscopy and analyzed automatically for nerve parameters. Additionally, the number and distribution of penetration sites was manually ascertained and the average length of the axons exiting them was computed. RESULTS: The earliest detectable changes included a significant increase in nerve density (6.06 ± 0.41% vs 8.98 ± 1.99%, P = 0.03) and branching (2867.8 ± 271.3/mm2 vs 4912.1 ± 1475.3/mm2 , P = 0.03), and in the number of penetration sites (258.80 ± 20.87 vs 422.60 ± 63.76, P = 0.0002) at 8 weeks of age. At 16 weeks, corneal innervation decreased, most notably in the periphery. The number of penetration sites remained significantly elevated relative to controls throughout the monitoring period. Similarly, prediabetic mice exhibited an increased number of penetration sites (242.2 ± 13.55 vs 305.6 ± 30.96, P = 0.003) without significant changes to the nerves. CONCLUSIONS: Our data suggest that diabetic peripheral neuropathy may be preceded by a phase of neuron growth rather than regression, and that the peripheral cornea is more sensitive than the center for detecting changes in innervation.