Severe communication delays are independent of seizure burden and persist despite contemporary treatments in SCN1A+ Dravet syndrome: Insights from the ENVISION natural history study.

OBJECTIVE: Dravet syndrome (DS) is a developmental and epileptic encephalopathy characterized by high seizure burden, treatment-resistant epilepsy, and developmental stagnation. Family members rate communication deficits among the most impactful disease manifestations. We evaluated seizure burden and language/communication development in children with DS. METHODS: ENVISION was a prospective, observational study evaluating children with DS associated with SCN1A pathogenic variants (SCN1A+ DS) enrolled at age ≤5 years. Seizure burden and antiseizure medications were assessed every 3 months and communication and language every 6 months with the Bayley Scales of Infant and Toddler Development 3rd edition and the parent-reported Vineland Adaptive Behavior Scales 3rd edition. We report data from the first year of observation, including analyses stratified by age at Baseline: 0:6-2:0 years:months (Y:M; youngest), 2:1-3:6 Y:M (middle), and 3:7-5:0 Y:M (oldest). RESULTS: Between December 2020 and March 2023, 58 children with DS enrolled at 16 sites internationally. Median follow-up was 17.5 months (range = .0-24.0), with 54 of 58 (93.1%) followed for at least 6 months and 51 of 58 (87.9%) for 12 months. Monthly countable seizure frequency (MCSF) increased with age (median [minimum-maximum] = 1.0 in the youngest [1.0-70.0] and middle [1.0-242.0] age groups and 4.5 [.0-2647.0] in the oldest age group), and remained high, despite use of currently approved antiseizure medications. Language/communication delays were observed early, and developmental stagnation occurred after age 2 years with both instruments. In predictive modeling, chronologic age was the only significant covariate of seizure frequency (effect size = .52, p = .024). MCSF, number of antiseizure medications, age at first seizure, and convulsive status epilepticus were not predictors of language/communication raw scores. SIGNIFICANCE: In infants and young children with SCN1A+ DS, language/communication delay and stagnation were independent of seizure burden. Our findings emphasize that the optimal therapeutic window to prevent language/communication delay is before 3 years of age.

NRF2 loss recapitulates heritable impacts of paternal cigarette smoke exposure.

Paternal cigarette smoke (CS) exposure is associated with increased risk of behavioral disorders and cancer in offspring, but the mechanism has not been identified. Here we use mouse models to investigate mechanisms and impacts of paternal CS exposure. We demonstrate that CS exposure induces sperm DNAme changes that are partially corrected within 28 days of removal from CS exposure. Additionally, paternal smoking is associated with changes in prefrontal cortex DNAme and gene expression patterns in offspring. Remarkably, the epigenetic and transcriptional effects of CS exposure that we observed in wild type mice are partially recapitulated in Nrf2-/- mice and their offspring, independent of smoking status. Nrf2 is a central regulator of antioxidant gene transcription, and mice lacking Nrf2 consequently display elevated oxidative stress, suggesting that oxidative stress may underlie CS-induced heritable epigenetic changes. Importantly, paternal sperm DNAme changes do not overlap with DNAme changes measured in offspring prefrontal cortex, indicating that the observed DNAme changes in sperm are not directly inherited. Additionally, the changes in sperm DNAme associated with CS exposure were not observed in sperm of unexposed offspring, suggesting the effects are likely not maintained across multiple generations.

The clinical, economic, and humanistic burden of Dravet syndrome - A systematic literature review.

Dravet syndrome (DS) is a developmental and epileptic encephalopathy with evolving disease course as individuals age. In recent years, the treatment landscape of DS has changed considerably, and a comprehensive systematic review of the contemporary literature is lacking. Here we synthesized published evidence on the occurrence of clinical impacts by age, the economic and humanistic (health-related quality-of-life [HRQoL]) burden, and health state utility. We provide an evidence-based, contemporary visualization of the clinical manifestations, highlighting that DS is not limited to seizures; non-seizure manifestations appear early in life and increase over time, contributing significantly to the economic and humanistic burden of disease. The primary drivers of HRQoL in DS include seizure severity, cognition, and motor and behavioral problems; in turn, these directly affect caregivers through the extent of assistance required and consequent impact on activities of daily living. Unsurprisingly, costs are driven by seizure-related events, hospitalizations, and in-home medical care visits. This systematic review highlights a paucity of longitudinal data; most studies meeting inclusion criteria were cross-sectional or had short follow-up. Nonetheless, available data illustrate the substantial impact on individuals, their families, and healthcare systems and establish the need for novel therapies to address the complex spectrum of DS manifestations.

Metabolic Alterations in Cellular Senescence: The Role of Citrate in Ageing and Age-Related Disease.

Recent mouse model experiments support an instrumental role for senescent cells in age-related diseases and senescent cells may be causal to certain age-related pathologies. A strongly supported hypothesis is that extranuclear chromatin is recognized by the cyclic GMP-AMP synthase-stimulator of interferon genes pathway, which in turn leads to the induction of several inflammatory cytokines as part of the senescence-associated secretory phenotype. This sterile inflammation increases with chronological age and age-associated disease. More recently, several intracellular and extracellular metabolic changes have been described in senescent cells but it is not clear whether any of them have functional significance. In this review, we highlight the potential effect of dietary and age-related metabolites in the modulation of the senescent phenotype in addition to discussing how experimental conditions may influence senescent cell metabolism, especially that of energy regulation. Finally, as extracellular citrate accumulates following certain types of senescence, we focus on the recently reported role of extracellular citrate in aging and age-related pathologies. We propose that citrate may be an active component of the senescence-associated secretory phenotype and via its intake through the diet may even contribute to the cause of age-related disease.

Safety and immunogenicity of a mosquito saliva peptide-based vaccine: a randomised, placebo-controlled, double-blind, phase 1 trial.

BACKGROUND: In animal models, immunity to mosquito salivary proteins protects animals against mosquito-borne disease. These findings provide a rationale to vaccinate against mosquito saliva instead of the pathogen itself. To our knowledge, no vector salivary protein-based vaccine has been tested for safety and immunogenicity in humans. We aimed to assess the safety and immunogenicity of Anopheles gambiae saliva vaccine (AGS-v), a peptide-based vaccine derived from four A gambiae salivary proteins, in humans. METHODS: In this randomised, placebo-controlled, double-blind, phase 1 trial, participants were enrolled at the National Institutes of Health Clinical Center in Bethesda, MD, USA. Participants were eligible if they were healthy adults, aged 18-50 years with no history of severe allergic reactions to mosquito bites. Participants were randomly assigned (1:1:1), using block randomisation and a computer-generated randomisation sequence, to treatment with either 200 nmol of AGS-v vaccine alone, 200 nmol of AGS-v with adjuvant (Montanide ISA 51), or sterile water as placebo. Participants and clinicians were masked to treatment assignment. Participants were given a subcutaneous injection of their allocated treatment at day 0 and day 21, followed by exposure to feeding by an uninfected Aedes aegypti mosquito at day 42 to assess subsequent risk to mosquito bites in a controlled setting. The primary endpoints were safety and immunogenicity at day 42 after the first immunisation. Participants who were given at least one dose of assigned treatment were assessed for the primary endpoints and analysis was by intention to treat. The trial was registered with ClinicalTrials.gov, NCT03055000, and is closed for accrual. FINDINGS: Between Feb 15 and Sept 10, 2017, we enrolled and randomly assigned 49 healthy adult participants to the adjuvanted vaccine (n=17), vaccine alone (n=16), or placebo group (n=16). Five participants did not complete the two-injection regimen with mosquito feeding at day 42, but were included in the safety analyses. No systemic safety concerns were identified; however, one participant in the adjuvanted vaccine group developed a grade 3 erythematous rash at the injection site. Pain, swelling, erythema, and itching were the most commonly reported local symptoms and were significantly increased in the adjuvanted vaccine group compared with both other treatment groups (nine [53%] of 17 participants in the adjuvanted vaccine group, two [13%] of 16 in the vaccine only group, and one [6%] of 16 in the placebo group; p=0·004). By day 42, participants who were given the adjuvanted vaccine had a significant increase in vaccine-specific total IgG antibodies compared with at baseline than did participants who were give vaccine only (absolute difference of log10-fold change of 0·64 [95% CI 0·39 to 0·89]; p=0·0002) and who were given placebo (0·62 [0·34 to 0·91]; p=0·0001). We saw a significant increase in IFN-γ production by peripheral blood mononuclear cells at day 42 in the adjuvanted vaccine group compared with in the placebo group (absolute difference of log10 ratio of vaccine peptide-stimulated vs negative control 0·17 [95% CI 0·061 to 0·27]; p=0·009) but we saw no difference between the IFN-γ production in the vaccine only group compared with the placebo group (0·022 [-0·072 to 0·116]; p=0·63). INTERPRETATION: AGS-v was well tolerated, and, when adjuvanted, immunogenic. These findings suggest that vector-targeted vaccine administration in humans is safe and could be a viable option for the increasing burden of vector-borne disease. FUNDING: Office of the Director and the Division of Intramural Research at the National Institute of Allergy and Infectious Diseases, and National Institutes of Health.

DRAVET ENGAGE. Parent caregivers of children with Dravet syndrome: Perspectives, needs, and opportunities for clinical research.

Dravet syndrome (DS) is an intractable developmental and epileptic encephalopathy significantly impacting affected children and their families. A novel, one-time, adeno-associated virus (AAV)-mediated gene regulation therapy was designed to treat the underlying cause of DS, potentially improving the full spectrum of DS manifestations. To ensure the first-in-human clinical trial addresses meaningful outcomes for patients and families, we examined their perspectives, priorities, goals, and desired outcomes in the design phase through a mixed methods approach (quantitative and qualitative). We conducted a non-identifiable parent caregiver survey, shared through a patient advocacy organization (n = 36 parents; children age ≤6 years). Parents were also engaged via three group discussions (n = 10; children age 2-20 years) and optional follow-up in-depth individual interviews (n = 6). Qualitative data analysis followed an inductive interpretive process, and qualitative researchers conducted a thematic analysis with a narrative approach. Survey results revealed most children (94%) were diagnosed by age 1, with onset of seizures at mean age 6.2 months and other DS manifestations before 2 years. The most desired disease aspects to address with potential new disease-modifying therapies were severe seizures (ranked by 92% of caregivers) and communication issues (development, expressive, receptive; 72-83%). Qualitative results showed the need for trial outcomes that recognize the impact of DS on the whole family. Parents eventually hope for trials including children of all ages and were both excited about the potential positive impact of a one-time disease-modifying therapy and mindful of potential long-term implications. Participants reflected on the details and risks of a clinical trial design (e.g., sham procedures) and described the different factors that relate to their decision to participate in a trial. Their main aspirations were to stop neurodevelopmental stagnation, to reduce seizures, and to reduce the impact on their families' wellbeing. To our knowledge, this is the first study within a patient-oriented research framework that specifically explored parents' needs and perceptions regarding clinical trials of a potential disease-modifying therapy for children with a severe, developmental disease, such as DS.

Timing storytime to maximize children's ability to retain new vocabulary.

Shared storybook reading is a key aid to vocabulary acquisition during childhood. However, word learning research has tended to use unnaturalistic (explicit) training regimes. Using a storybook paradigm, we examined whether children (particularly those with weaker vocabularies) are more likely to retain new words if they learn them closer to sleep. Parents read their children (5- to 7-year-olds; N = 237) an alien adventure story that contained 12 novel words with illustrations at one of two training times: at bedtime or 3-5 h before bedtime. Using online tasks, parents tested their children's ability to recall the new words (production) and associate them with pictures (comprehension) immediately after hearing the story and again the following morning. As hypothesized, we replicated two findings. First, children showed overnight improvements in their ability to produce and comprehend new words when tested again the next day. Second, children with better existing vocabulary knowledge showed larger overnight gains in new word comprehension. Counter to expectations, overnight gains in comprehension were larger if the story was read 3-5 h before bedtime rather than at bedtime. These ecologically valid findings are consistent with theories that characterize word learning as a prolonged process supported by mechanisms such as consolidation and retrieval practice, with existing vocabulary knowledge acting as an important source of variability in retention. The findings provide preliminary evidence that encountering new words in stories later in the day (but not too close to sleep) may help to harness vocabulary growth and may be more beneficial than leaving shared storybook reading just for bedtime.

Immunogenicity, Safety, and Efficacy of a Standalone Universal Influenza Vaccine, FLU-v, in Healthy Adults: A Randomized Clinical Trial.

Background: FLU-v is a broad-spectrum influenza vaccine that induces antibodies and cell-mediated immunity. Objective: To compare the safety, immunogenicity, and exploratory efficacy of different formulations and dosing regimens of FLU-v versus placebo. Design: Randomized, double-blind, placebo-controlled, single-center phase 2b clinical trial. (ClinicalTrials.gov: NCT02962908; EudraCT: 2015-001932-38). Setting: The Netherlands. Participants: 175 healthy adults aged 18 to 60 years. Intervention: 0.5-mL subcutaneous injection of 500 µg of adjuvanted (1 dose) or nonadjuvanted (2 doses) FLU-v (A-FLU-v or NA-FLU-v) or adjuvanted or nonadjuvanted placebo (A-placebo or NA-placebo) (2:2:1:1 ratio). Measurements: Vaccine-specific cellular responses at days 0, 42, and 180 were assessed via flow cytometry and enzyme-linked immunosorbent assay. Solicited information on adverse events (AEs) was collected for 21 days after vaccination. Unsolicited information on AEs was collected throughout the study. Results: The AEs with the highest incidence were mild to moderate injection site reactions. The difference between A-FLU-v and A-placebo in the median fold increase in secreted interferon-γ (IFN-γ) was 38.2-fold (95% CI, 4.7- to 69.7-fold; P = 0.001) at day 42 and 25.0-fold (CI, 5.7- to 50.9-fold; P < 0.001) at day 180. The differences between A-FLU-v and A-placebo in median fold increase at day 42 were 4.5-fold (CI, 2.3- to 9.8-fold; P < 0.001) for IFN-γ-producing CD4+ T cells, 4.9-fold (CI, 1.3- to 40.0-fold; P < 0.001) for tumor necrosis factor-α (TNF-α), 7.0-fold (CI, 3.5- to 18.0-fold; P < 0.001) for interleukin-2 (IL-2), and 1.7-fold (CI, 0.1- to 4.0-fold; P = 0.004) for CD107a. At day 180, differences were 2.1-fold (CI, 0.0- to 6.0-fold; P = 0.030) for IFN-γ and 5.7-fold (CI, 2.0- to 15.0-fold; P < 0.001) for IL-2, with no difference for TNF-α or CD107a. No differences were seen between NA-FLU-v and NA-placebo. Limitation: The study was not powered to evaluate vaccine efficacy against influenza infection. Conclusion: Adjuvanted FLU-v is immunogenic and merits phase 3 development to explore efficacy. Primary Funding Source: SEEK and the European Commission Directorate-General for Research and Innovation, European Member States within the UNISEC (Universal Influenza Vaccines Secured) project.

Bioengineering Clinically Relevant Cardiomyocytes and Cardiac Tissues from Pluripotent Stem Cells.

The regenerative capacity of cardiomyocytes is insufficient to functionally recover damaged tissue, and as such, ischaemic heart disease forms the largest proportion of cardiovascular associated deaths. Human-induced pluripotent stem cells (hiPSCs) have enormous potential for developing patient specific cardiomyocytes for modelling heart disease, patient-based cardiac toxicity testing and potentially replacement therapy. However, traditional protocols for hiPSC-derived cardiomyocytes yield mixed populations of atrial, ventricular and nodal-like cells with immature cardiac properties. New insights gleaned from embryonic heart development have progressed the precise production of subtype-specific hiPSC-derived cardiomyocytes; however, their physiological immaturity severely limits their utility as model systems and their use for drug screening and cell therapy. The long-entrenched challenges in this field are being addressed by innovative bioengingeering technologies that incorporate biophysical, biochemical and more recently biomimetic electrical cues, with the latter having the potential to be used to both direct hiPSC differentiation and augment maturation and the function of derived cardiomyocytes and cardiac tissues by mimicking endogenous electric fields.

Sleep-dependent consolidation in children with comprehension and vocabulary weaknesses: it'll be alright on the night?

BACKGROUND: Vocabulary is crucial for an array of life outcomes and is frequently impaired in developmental disorders. Notably, 'poor comprehenders' (children with reading comprehension deficits but intact word reading) often have vocabulary deficits, but underlying mechanisms remain unclear. Prior research suggests intact encoding but difficulties consolidating new word knowledge. We test the hypothesis that poor comprehenders' sleep-associated vocabulary consolidation is compromised by their impoverished lexical-semantic knowledge. METHODS: Memory for new words was tracked across wake and sleep to assess encoding and consolidation in 8-to-12-year-old good and poor comprehenders. Each child participated in two sets of sessions, one beginning in the morning (AM-encoding) and the other in the evening (PM-encoding). In each case, they were taught 12 words and were trained on a spatial memory task. Memory was assessed immediately, 12- and 24-hr later via stem-completion, picture-naming, and definition tasks to probe different aspects of word knowledge. Long-term retention was assessed 1-2 months later. RESULTS: Recall of word-forms improved over sleep and postsleep wake, as measured in both stem-completion and picture-naming tasks. Counter to hypotheses, deficits for poor comprehenders were not observed in consolidation but instead were seen across measures and throughout testing, suggesting a deficit from encoding. Variability in vocabulary knowledge across the whole sample predicted sleep-associated consolidation, but only when words were learned early in the day and not when sleep followed soon after learning. CONCLUSIONS: Poor comprehenders showed weaker memory for new words than good comprehenders, but sleep-associated consolidation benefits were comparable between groups. Sleeping soon after learning had long-lasting benefits for memory and may be especially beneficial for children with weaker vocabulary. These results provide new insights into the breadth of poor comprehenders' vocabulary weaknesses, and ways in which learning might be better timed to remediate vocabulary difficulties.

Disease burden of Crigler-Najjar syndrome: Systematic review and future perspectives.

BACKGROUND AND AIM: Crigler-Najjar syndrome (CNS) results from biallelic mutations of UGT1A1 causing partial or total loss of uridine 5'-diphosphate glucuronyltransferase activity leading to unconjugated hyperbilirubinemia and its attendant risk for irreversible neurological injury (kernicterus). CNS is exceedingly rare and has been only partially characterized through relatively small studies, each comprising between two and 57 patients. METHODS: A systematic literature review was conducted to consolidate data on the patient, caregiver, and societal burden of CNS. RESULTS: Twenty-eight articles on clinical aspects of CNS were identified, but no published data on its humanistic or economic burden were found. In patients with complete UGT1A1 deficiency (type 1 CNS [CNS-I]), unconjugated bilirubin levels increase 3-6 mg/dL/day during the newborn period and reach neurologically dangerous levels between 5 and 14 days of age. Phototherapy is the mainstay of treatment but poses significant challenges to patients and their families. Despite consistent phototherapy, patients with CNS-I have worsening hyperbilirubinemia with advancing age. Liver transplantation is the only definitive therapy for CNS-I and is increasingly associated with excellent long-term survival but also incurs high costs, medical and surgical morbidities, and risks of immunosuppression. CONCLUSIONS: Crigler-Najjar syndrome is associated with a substantial burden, even with existing standards of care. The development of novel disease-modifying therapies has the potential to reduce disease burden and improve the lives of CNS patients and their families.

The Role of the Epididymis and the Contribution of Epididymosomes to Mammalian Reproduction.

It is well-established that testicular spermatozoa are immature and acquire motility and fertilization capabilities during transit throughout the epididymis. The epididymis is a duct-like organ that connects the testis to the vas deferens and is comprised of four anatomical regions: the initial segment, caput, corpus, and cauda. Sperm maturation occurs during epididymal transit by the interaction of sperm cells with the unique luminal environment of each epididymal region. In this review we discuss the epididymis as an essential reproductive organ responsible for sperm concentration, maturation (including sperm motility acquisition and fertilizing ability), protection and storage. Importantly, we also discuss specific characteristics and roles of epididymal-derived exosomes (epididymosomes) in establishing sperm competency within the intricate process of reproduction. This review suggests that an increasing body of evidence is working to develop a complete picture of the role of the epididymis in male reproduction, offspring health, and disease susceptibility.

Offline consolidation supersedes prior knowledge benefits in children's (but not adults') word learning.

Prior linguistic knowledge is proposed to support the acquisition and consolidation of new words. Adults typically have larger vocabularies to support word learning than children, but the developing brain shows enhanced neural processes that are associated with offline memory consolidation. This study investigated contributions of prior knowledge to initial word acquisition and consolidation at different points in development, by teaching children and adults novel words (e.g., ballow) that varied in the number of English word-form "neighbours" (e.g., wallow, bellow). Memory for the novel word-forms was tested immediately after training, the next day and 1 week later, to assess the time-course of prior knowledge contributions. Children aged 7-9 years (Experiments 1, 3) and adults (Experiment 2) recalled words with neighbours better than words without neighbours when tested immediately after training. However, a period of offline consolidation improved overall recall and reduced the influence of word-form neighbours on longer term memory. These offline consolidation benefits were larger in children than adults, supporting theories that children have a greater propensity for consolidating phonologically distinctive language information. Local knowledge of just a single word-form neighbour was enough to enhance learning, and this led to the individual differences in word recall that were related to adults' global vocabulary ability. The results support the proposal that the relative contributions of different learning mechanisms change across the lifespan, and highlight the importance of testing theoretical models of word learning in the context of development.

A multicenter, retrospective medical record review of X-linked myotubular myopathy: The recensus study.

INTRODUCTION: X-linked myotubular myopathy (XLMTM), characterized by severe hypotonia, weakness, respiratory distress, and early mortality, is rare and natural history studies are few. METHODS: RECENSUS is a multicenter chart review of male XLMTM patients characterizing disease burden and unmet medical needs. Data were collected between September 2014 and June 2016. RESULTS: Analysis included 112 patients at six clinical sites. Most recent patient age recorded was ≤18 months for 40 patients and >18 months for 72 patients. Mean (SD) age at diagnosis was 3.7 (3.7) months and 54.3 (77.1) months, respectively. Mortality was 44% (64% ≤18 months; 32% >18 months). Premature delivery occurred in 34/110 (31%) births. Nearly all patients (90%) required respiratory support at birth. In the first year of life, patients underwent an average of 3.7 surgeries and spent 35% of the year in the hospital. DISCUSSION: XLMTM is associated with high mortality, disease burden, and healthcare utilization. Muscle Nerve 57: 550-560, 2018.

The humanistic burden of Pompe disease: are there still unmet needs? A systematic review.

BACKGROUND: Humanistic burden considers the impact of an illness on a patient's health-related quality of life (HRQoL), activities of daily living (ADL), caregiver health, and caregiver QoL. Humanistic burden also considers treatment satisfaction and adherence to treatment regimens. Pompe disease is an autosomal recessive, progressive, multisystemic neuromuscular disease. Approval of enzyme-replacement therapy (ERT) markedly improved prognosis for patients, but considerable morbidity and a substantial humanistic burden remain. This article characterizes the humanistic burden of Pompe disease through a systematic literature review. METHODS: A systematic search of MEDLINE® and Embase® with back-referencing and supplementary literature searches was performed to retrieve data from interventional and non-interventional studies on the humanistic burden of Pompe disease. Publications were screened according to predefined criteria, extracted, and assessed for quality. Extracted data were narratively synthesized. RESULTS: No publications on the humanistic burden of infantile-onset Pompe disease (IOPD) were identified. As such, of 17 publications included here, all are in patients with late-onset Pompe disease (LOPD). Thirteen publications were initiated after approval of ERT, two were initiated before, and two overlapped the approval of ERT. The review shows that LOPD patients have a significantly lower HRQoL than the general population, even if treated with ERT. On transitioning to ERT, treatment was associated with improvement in the physical component score of the SF-36 and fatigue, although the SF-36 mental component score remained stable. Physical HRQoL remained below population norms after 4 years of ERT. Significantly more ERT-treated patients reported pain than controls, and bodily pain worsened in later years following ERT initiation. Treatment-naïve LOPD patients had significantly poorer ADL functioning compared with the general population, although ERT stabilized deteriorating functioning impairment. ERT studies showed caregivers provide 17.7 h/week informal care on average. Fifty percent, 40% and <20% of caregivers reported mental health, physical health, and financial/relational problems, respectively. In ERT-naïve patients, wheelchair use and home ventilatory support was associated with lower physical HRQoL and ADL functioning. In ERT-treated patients, key factors predicting worse HRQoL and ADL functioning were higher respiratory distress, poorer sleep quality, greater pain, and more fatigue. CONCLUSIONS: Pompe disease has a substantial humanistic burden, with strong inter-relationships among and between humanistic burden parameters and clinical progression.

Areca nut alkaloids induce irreparable DNA damage and senescence in fibroblasts and may create a favourable environment for tumour progression.

BACKGROUND: Oral submucous fibrosis (OSMF) is a pre-malignant condition that is strongly associated with the areca nut alkaloids, arecoline (ARC) and arecaidine (ARD). The condition is characterised by the presence of senescent fibroblasts in the subepithelial mesenchyme which have the potential to promote malignancy in the neighbouring epithelial cells. We tested the hypothesis that areca nut alkaloids induce senescence in oral fibroblasts and promote the secretion of invasion-promoting transforming growth factor ß (TGF-ß) and matrix metalloproteinase-2 (MMP-2). METHODS: Two oral fibroblast lines were treated for 48h with ARC and ARD. Senescence-associated ß-galactosidase (SA-ßGal) activity, Ki67 (cycling cells), large 53BP1 foci (irreparable DNA strand breaks) and p16(INK) (4A) (late senescence) were used as markers of cellular senescence and were quantified using indirect immunofluorescence and the ImageJ program. TGF-ß and MMP-2 levels were measured using ELISA. Statistical analyses were performed with the two-tailed unpaired t-test where n = 3 and the Wilcoxon-Mann-Whitney test where n = 6. RESULTS: ARC (100 and 300 µM) and ARD (30 and 100 µM) significantly (P < 0.05) induced fibroblast senescence, as determined by the increased expression of SA-ßGal, 53BP1 staining and CDKN2A/p16(INK) (4A) ; there was also a non-significant reduction in Ki67 staining. Treated cells also showed a three- fivefold increase in TGF-ß and a small non-significant increase in MMP-2. CONCLUSIONS: Areca nut alkaloids induce senescence in oral fibroblasts and promote increased secretion of TGF-ß and perhaps MMP-2 that may create a tissue environment thought to be critical in the progression of OSMF to malignancy.

Senescence-Derived Extracellular Molecules as Modulators of Oral Cancer Development: A Mini-Review.

Oral cancers are predominantly oral squamous cell carcinomas (OSCCs) derived from keratinocytes, and there is now very detailed knowledge of the genetics and molecular biology of the epithelial tumourigenic component of these cancers, including the identification of cancer stem or tumour-initiating cells. Several key genetic alterations have been identified including the near ubiquitous loss of the CDKN2A/p16INK4A and p53 pathways and telomerase activation, together with frequent inactivation of the NOTCH1 canonical pathway either by somatic genetic alterations or by the presence of human papilloma virus. There is also evidence that OSCCs arise from a 'field' of altered cells and that malignant conversion takes place pre-dominantly at the microscopic level. However, in the last decade, it has been realised that tumour development and progression are influenced by the cells of the microenvironment with cross-talk between the epithelial (tumour) and mesenchymal components. OSCCs, especially those that have bypassed cellular senescence, produce an array of proteins and metabolites that induce cellular senescence in the normal surrounding cells; indeed, senescence is a common property of cancer-associated fibroblasts (CAFs). Cellular senescence is defined as an irreversible cell cycle arrest and is associated with the release of molecules known as the senescence-associated secretory phenotype that can selectively promote the growth of pre-neoplastic keratinocytes (osteopontin) and cancer invasion (transforming growth factor ß, matrix metalloproteinases, interleukin 6 and lactate). In addition, both old and new work has shown that keratinocytes harbouring NOTCH loss-of-function mutations that lead to defective keratinocyte differentiation and loss of squamous epithelial barrier function may act as a tumour-promoting stimulus for initiated cells harbouring RAS pathway mutations by activating a wound response in the tumour mesenchyme. Thus, not all keratinocytes in the tumour tissue may be tumourigenic and may instead act as promoters of tumour growth and progression analogous to the much-studied CAFs which co-evolve with the genetically altered tumourigenic cells. This new data is discussed in relation to attempts to develop novel non-invasive diagnostics and therapeutics for oral cancer.

Senescent human fibroblasts show increased glycolysis and redox homeostasis with extracellular metabolomes that overlap with those of irreparable DNA damage, aging, and disease.

Cellular senescence can modulate various pathologies and is associated with irreparable DNA double-strand breaks (IrrDSBs). Extracellular senescence metabolomes (ESMs) were generated from fibroblasts rendered senescent by proliferative exhaustion (PEsen) or 20 Gy of γ rays (IrrDSBsen) and compared with those of young proliferating cells, confluent cells, quiescent cells, and cells exposed to repairable levels of DNA damage to identify novel noninvasive markers of senescent cells. ESMs of PEsen and IrrDSBsen overlapped and showed increased levels of citrate, molecules involved in oxidative stress, a sterol, monohydroxylipids, tryptophan metabolism, phospholipid, and nucleotide catabolism, as well as reduced levels of dipeptides containing branched chain amino acids. The ESM overlaps with the aging and disease body fluid metabolomes, supporting their utility in the noninvasive detection of human senescent cells in vivo and by implication the detection of a variety of human pathologies. Intracellular metabolites of senescent cells showed a relative increase in glycolysis, gluconeogenesis, the pentose-phosphate pathway, and, consistent with this, pyruvate dehydrogenase kinase transcripts. In contrast, tricarboxylic acid cycle enzyme transcript levels were unchanged and their metabolites were depleted. These results are surprising because glycolysis antagonizes senescence entry but are consistent with established senescent cells entering a state of low oxidative stress.

Serum metabolomics in animal models and human disease.

PURPOSE OF REVIEW: The aim of this study is to highlight some recent uses of serum metabolomics in human and animal studies. The main themes are the importance of understanding the underlying variation in human metabolism and the use of serum metabolomics in disease profiling. RECENT FINDINGS: Several studies have attempted to use serum metabolomics to develop noninvasive biomarkers of disease and/or track the consequences of nutritional and genetic interventions. Many advances have been made with common changes being identified in ageing, the menopause and cancer but several problems of interpretation have emerged from these studies. These include the small sample sizes in most human studies and the differences between human and rodent metabolomes. However, a metabolic screen of over 1000 'healthy' humans (the Humsermet project) has highlighted many variables that may be used to refine the interpretation and design of previous and future human studies alike, in addition to data mining. SUMMARY: Some common serum metabolome alterations have been identified but many inconsistencies remain. The construction of a human serum metabolome database should be informative in the design of future human and animal model studies.

Heterozygous Eif4nif1 Stop Gain Mice Replicate the Primary Ovarian Insufficiency Phenotype in Women.

We created the c.1286C>G stop-gain mutation found in a family with primary ovarian insufficiency (POI) at age 30 years. The Eif4enif1 C57/Bl6 transgenic mouse model contained a floxed exon 10-19 cassette with a conditional knock-in cassette containing the c.1286C>G stop-gain mutation in exon 10. The hybrid offspring of CMV- Cre mice with Eif4enif1 WT/flx mice were designated Eif4enif1 WT/ Δ for simplicity. A subset of female heterozygotes ( Eif4enif1 WT/ Δ ) had no litters. In those with litters, the final litter was earlier (5.4±2.6 vs. 10.5±0.7 months; p=0.02). Heterozygous breeding pair ( Eif4enif1 WT/ Δ x Eif4enif1 WT/ Δ ) litter size was 60% of WT litter size (3.9±2.0 vs. 6.5±3.0 pups/litter; p <0.001). The genotypes were 35% Eif4enif1 WT/flx and 65% Eif4enif1 WT/ Δ , with no homozygotes. Homozygote embryos did not develop beyond the 4-8 cell stage. The number of follicles in ovaries from Eif4enif1 WT/ Δ mice was lower starting at the primordial (499±290 vs. 1445±381) and primary follicle stage (1069±346 vs. 1450±193) on day 10 (p<0.05). The preantral follicle number was lower starting on day 21 (213±86 vs. 522±227; p<0.01). Examination of ribosome protected mRNAs (RPR) demonstrated altered mRNA expression. The Eif4enif1 stop-gain mice replicate the POI phenotype in women. The unique mouse model provides a platform to study regulation of protein translation across oocyte and embryo development in mammals.