Astrocytic Insulin Signaling Couples Brain Glucose Uptake with Nutrient Availability.

We report that astrocytic insulin signaling co-regulates hypothalamic glucose sensing and systemic glucose metabolism. Postnatal ablation of insulin receptors (IRs) in glial fibrillary acidic protein (GFAP)-expressing cells affects hypothalamic astrocyte morphology, mitochondrial function, and circuit connectivity. Accordingly, astrocytic IR ablation reduces glucose-induced activation of hypothalamic pro-opio-melanocortin (POMC) neurons and impairs physiological responses to changes in glucose availability. Hypothalamus-specific knockout of astrocytic IRs, as well as postnatal ablation by targeting glutamate aspartate transporter (GLAST)-expressing cells, replicates such alterations. A normal response to altering directly CNS glucose levels in mice lacking astrocytic IRs indicates a role in glucose transport across the blood-brain barrier (BBB). This was confirmed in vivo in GFAP-IR KO mice by using positron emission tomography and glucose monitoring in cerebral spinal fluid. We conclude that insulin signaling in hypothalamic astrocytes co-controls CNS glucose sensing and systemic glucose metabolism via regulation of glucose uptake across the BBB.

Activation of endogenous retroviruses during brain development causes an inflammatory response.

Endogenous retroviruses (ERVs) make up a large fraction of mammalian genomes and are thought to contribute to human disease, including brain disorders. In the brain, aberrant activation of ERVs is a potential trigger for an inflammatory response, but mechanistic insight into this phenomenon remains lacking. Using CRISPR/Cas9-based gene disruption of the epigenetic co-repressor protein Trim28, we found a dynamic H3K9me3-dependent regulation of ERVs in proliferating neural progenitor cells (NPCs), but not in adult neurons. In vivo deletion of Trim28 in cortical NPCs during mouse brain development resulted in viable offspring expressing high levels of ERVs in excitatory neurons in the adult brain. Neuronal ERV expression was linked to activated microglia and the presence of ERV-derived proteins in aggregate-like structures. This study demonstrates that brain development is a critical period for the silencing of ERVs and provides causal in vivo evidence demonstrating that transcriptional activation of ERV in neurons results in an inflammatory response.

The centrosome protein AKNA regulates neurogenesis via microtubule organization.

The expansion of brain size is accompanied by a relative enlargement of the subventricular zone during development. Epithelial-like neural stem cells divide in the ventricular zone at the ventricles of the embryonic brain, self-renew and generate basal progenitors1 that delaminate and settle in the subventricular zone in enlarged brain regions2. The length of time that cells stay in the subventricular zone is essential for controlling further amplification and fate determination. Here we show that the interphase centrosome protein AKNA has a key role in this process. AKNA localizes at the subdistal appendages of the mother centriole in specific subtypes of neural stem cells, and in almost all basal progenitors. This protein is necessary and sufficient to organize centrosomal microtubules, and promote their nucleation and growth. These features of AKNA are important for mediating the delamination process in the formation of the subventricular zone. Moreover, AKNA regulates the exit from the subventricular zone, which reveals the pivotal role of centrosomal microtubule organization in enabling cells to both enter and remain in the subventricular zone. The epithelial-to-mesenchymal transition is also regulated by AKNA in other epithelial cells, demonstrating its general importance for the control of cell delamination.

Transposable Elements: A Common Feature of Neurodevelopmental and Neurodegenerative Disorders.

The etiology of most neurological disorders is poorly understood and current treatments are largely ineffective. New ideas and concepts are therefore vitally important for future research in this area. This review explores the concept that dysregulation of transposable elements (TEs) contributes to the appearance and pathology of neurodevelopmental and neurodegenerative disorders. Despite TEs making up at least half of the human genome, they are vastly understudied in relation to brain disorders. However, recent advances in sequencing technologies and gene editing approaches are now starting to unravel the pathological role of TEs. Aberrant activation of TEs has been found in many neurological disorders; the resulting pathogenic effects, which include alterations of gene expression, neuroinflammation, and direct neurotoxicity, are starting to be resolved. An increased understanding of the relationship between TEs and pathological processes in the brain improves the potential for novel diagnostics and interventions for brain disorders.

Distinct subcellular autophagy impairments in induced neurons from patients with Huntington's disease.

Huntington's disease is a neurodegenerative disorder caused by CAG expansions in the huntingtin (HTT) gene. Modelling Huntington's disease is challenging, as rodent and cellular models poorly recapitulate the disease as seen in ageing humans. To address this, we generated induced neurons through direct reprogramming of human skin fibroblasts, which retain age-dependent epigenetic characteristics. Huntington's disease induced neurons (HD-iNs) displayed profound deficits in autophagy, characterized by reduced transport of late autophagic structures from the neurites to the soma. These neurite-specific alterations in autophagy resulted in shorter, thinner and fewer neurites specifically in HD-iNs. CRISPRi-mediated silencing of HTT did not rescue this phenotype but rather resulted in additional autophagy alterations in control induced neurons, highlighting the importance of wild-type HTT in normal neuronal autophagy. In summary, our work identifies a distinct subcellular autophagy impairment in adult patient derived Huntington's disease neurons and provides a new rationale for future development of autophagy activation therapies.

Bacterial colonisation during regular daily use of a power-driven water flosser and risk for cross-contamination. Can it be prevented?

OBJECTIVE: To assess whether bacterial colonisation in a power-driven water flosser can be prevented. MATERIALS AND METHODS: Twenty-four patients undergoing supportive periodontal treatment used 2 power-driven water flossers [Sonicare AirFloss (SAF), AirFloss Ultra (SAFU)] for 12 weeks each as follows: (a) with bottled water (BW); (b) with BW and cleaning the device extra-orally twice per week with chlorhexidine gluconate or (c) essential-oil-based (EO) mouth-rinse; (d) with EO only. Water-jet samples were taken after 6 and 12 weeks with the used nozzle and after exchanging to a brand-new nozzle. After 12 weeks, all devices underwent an intensive cleaning procedure. Samples were analysed by PCR-based method for cariogenic and periodontal pathogens and culture for staphylococci, aerobe gram-negative bacteria, and Candida sp. RESULTS: Contamination of SAF/SAFU with Streptococcus mutans was found in > 95% of the samples; periodontal pathogens and aerobe gram-negative bacteria were detected in 19-56% of the samples, while Staphylococcus aureus and Candida sp. were identified only in few samples. Contamination rate was basically unaffected by time-point, device, or way of use. Further, exchanging the nozzle did not prevent transmission of a contaminated water-jet, but the intensive cleaning reduced most of the pathogens significantly, except of S. mutans. CONCLUSION: Neither a specific way of use nor exchanging the nozzle prevented bacterial colonisation and transmission of biofilm components via the water-jet of SAF/SAFU. CLINICAL RELEVANCE: Bacterial colonisation in a power-driven water flosser seems impossible to prevent; to restrict the risk of cross-contamination within a household, one device per person should be recommended.

Organisational models of health services for children and adolescents in out-of-home care: Health technology assessment.

AIM: Decades of research confirm that children and adolescents in out-of-home care (foster family, residential care) have much greater health care needs than their peers. A systematic literature review was conducted to evaluate organisational health care models for this vulnerable group. METHODS: A systematic literature search was undertaken of the following databases: Academic Search Elite, CENTRAL, Cochrane Database of Systematic Reviews, Cinahl, DARE, ERIC, HTA, PsycInfo, Psychology and Behavioural Sciences Collection, PubMed, SocIndex. Randomised and non-randomised controlled trials were to be included. Two pairs of reviewers independently assessed abstracts of the identified published papers. Abstracts meeting the inclusion criteria were ordered in full text. Each article was reviewed independently, by pairs of reviewers. A joint assessment was made based on the inclusion criteria and relevance. Cases of disagreement were resolved by consensus discussion. RESULTS: No study with low or medium risk of bias was identified. CONCLUSION: In the absence of studies of acceptable quality, it is not possible to assess the impact of organisational models intended to ensure adequate health and dental care for children and adolescents in out-of-home care. Therefore, well-designed follow-up studies should be conducted following the implementation of such models.

The transcription factor Otx2 regulates choroid plexus development and function.

The choroid plexuses (ChPs) are the main regulators of cerebrospinal fluid (CSF) composition and thereby also control the composition of a principal source of signaling molecules that is in direct contact with neural stem cells in the developing brain. The regulators of ChP development mediating the acquisition of a fate that differs from the neighboring neuroepithelial cells are poorly understood. Here, we demonstrate in mice a crucial role for the transcription factor Otx2 in the development and maintenance of ChP cells. Deletion of Otx2 by the Otx2-CreERT2 driver line at E9 resulted in a lack of all ChPs, whereas deletion by the Gdf7-Cre driver line affected predominately the hindbrain ChP, which was reduced in size, primarily owing to an increase in apoptosis upon Otx2 deletion. Strikingly, Otx2 was still required for the maintenance of hindbrain ChP cells at later stages when Otx2 deletion was induced at E15, demonstrating a central role of Otx2 in ChP development and maintenance. Moreover, the predominant defects in the hindbrain ChP mediated by Gdf7-Cre deletion of Otx2 revealed its key role in regulating early CSF composition, which was altered in protein content, including the levels of Wnt4 and the Wnt modulator Tgm2. Accordingly, proliferation and Wnt signaling levels were increased in the distant cerebral cortex, suggesting a role of the hindbrain ChP in regulating CSF composition, including key signaling molecules. Thus, Otx2 acts as a master regulator of ChP development, thereby influencing one of the principal sources of signaling in the developing brain, the CSF.

Mini-heterochromatin domains constrain the cis-regulatory impact of SVA transposons in human brain development and disease.

SVA (SINE (short interspersed nuclear element)-VNTR (variable number of tandem repeats)-Alu) retrotransposons remain active in humans and contribute to individual genetic variation. Polymorphic SVA alleles harbor gene regulatory potential and can cause genetic disease. However, how SVA insertions are controlled and functionally impact human disease is unknown. Here we dissect the epigenetic regulation and influence of SVAs in cellular models of X-linked dystonia parkinsonism (XDP), a neurodegenerative disorder caused by an SVA insertion at the TAF1 locus. We demonstrate that the KRAB zinc finger protein ZNF91 establishes H3K9me3 and DNA methylation over SVAs, including polymorphic alleles, in human neural progenitor cells. The resulting mini-heterochromatin domains attenuate the cis-regulatory impact of SVAs. This is critical for XDP pathology; removal of local heterochromatin severely aggravates the XDP molecular phenotype, resulting in increased TAF1 intron retention and reduced expression. Our results provide unique mechanistic insights into how human polymorphic transposon insertions are recognized and how their regulatory impact is constrained by an innate epigenetic defense system.

Comparison of the cost-effectiveness of a high- and a low-intensity smoking cessation intervention in Sweden: a randomized trial.

OBJECTIVE: To assess the relative cost-effectiveness of a high-intensity treatment (HIT) and a low-intensity treatment (LIT) for smoking cessation. METHODS: The societal and health care perspective economic evaluation was based on the reported number of quitters at 12-month follow-up (point prevalence) from a randomized controlled trial of 2 smoking cessation programs in Sweden. Future disease-related costs (in Swedish kronor [SEK] 2004; SEK7.35 = USD1) and health effects (in quality-adjusted life-years [QALYs]) were estimated via a Markov model comprising lung cancer, chronic obstructive pulmonary disease, and cardiovascular disease including stroke with costs and QALYs discounted 3% annually. RESULTS: HIT was more effective than LIT (23% vs. 16% quitters), but at a considerably higher intervention cost: SEK26,100 versus 9,100 per quitter. The model-estimated societal costs avoided did not balance the higher intervention costs, so the incremental cost-effectiveness ratio (ICER) amounted to SEK100,000 per QALY for HIT versus LIT. All sensitivity analyses indicated an ICER below SEK300,000 and that HIT is the preferred option if the decision maker willingness-to-pay exceeds SEK50,000 per QALY. Compared with no intervention, LIT was cost saving, whereas HIT was estimated at SEK8,400 per QALY. CONCLUSIONS: Compared with no smoking cessation program, it is a societal waste not to implement the LIT as it is estimated to result in lower societal costs. The incremental cost per QALY gained of SEK100,000 for HIT is considered very cost-effective in Sweden. Thus, if smoking cessation programs are judged in the same manner as other Swedish health care measures, the high-intensity program should be chosen before the low-cost program.

Heterogeneity in cost-effectiveness of lifestyle counseling for metabolic syndrome risk groups -primary care patients in Sweden.

BACKGROUND: Clinical trials have indicated that lifestyle interventions for patients with lifestyle-related cardiovascular and diabetes risk factors (the metabolic syndrome) are cost-effective. However, patient characteristics in primary care practice vary considerably, i.e. they exhibit heterogeneity in risk factors. The cost-effectiveness of lifestyle interventions is likely to differ over heterogeneous patient groups. METHODS: Patients (62 men, 80 women) in the Kalmar Metabolic Syndrome Program (KMSP) in primary care (Kalmar regional healthcare area, Sweden) were divided into three groups reflecting different profiles of metabolic risk factors (low, middle and high risk) and gender. A Markov model was used to predict future cardiovascular disease and diabetes, including complications (until age 85 years or death), with health effects measured as QALYs and costs from a societal perspective in Euro (EUR) 2012, discounted 3%. Simulations with risk factor levels at start and at 12 months follow-up were performed for each group, with an assumed 4-year sustainability of intervention effects. RESULTS: The program was estimated cost-saving for middle and high risk men, while the incremental cost vs. do-nothing varied between EUR 3,500 - 18,000 per QALY for other groups. There is heterogeneity in the cost-effectiveness over the risk groups but this does not affect the overall conclusion on the cost-effectiveness of the KMSP. Even the highest ICER (for high risk women) is considered moderately cost-effective in Sweden. The base case result was not sensitive to alternative data and methodology but considerably affected by sustainability assumptions. Alternative risk stratifications did not change the overall conclusion that KMSP is cost-effective. However, simple grouping with average risk factor levels over gender groups overestimate the cost-effectiveness. CONCLUSIONS: Lifestyle counseling to prevent metabolic diseases is cost-effective in Swedish standard primary care settings. The use of risk stratification in the cost-effectiveness analysis established that the program was cost-effective for all patient groups, even for those with very high levels of lifestyle-related risk factors for the metabolic syndrome diseases. Heterogeneity in the cost-effectiveness of lifestyle interventions in primary care patients is expected, and should be considered in health policy decisions.

HPV self-sampling versus healthcare provider collection on the effect of cervical cancer screening uptake and costs in LMIC: a systematic review and meta-analysis.

BACKGROUND: Cervical cancer is a major global health issue, with 89% of cases occurring in low- and middle-income countries (LMICs). Human papillomavirus (HPV) self-sampling tests have been suggested as an innovative way to improve cervical cancer screening uptake and reduce the burden of disease. The objective of this review was to examine the effect of HPV self-sampling on screening uptake compared to any healthcare provider sampling in LMICs. The secondary objective was to estimate the associated costs of the various screening methods. METHOD: Studies were retrieved from PubMed, Embase, CINAHL, CENTRAL (by Cochrane), Web of Science, and ClinicalTrials.gov up until April 14, 2022, and a total of six trials were included in the review. Meta-analyses were performed mainly using the inverse variance method, by pooling effect estimates of the proportion of women who accepted the screening method offered. Subgroup analyses were done comparing low- and middle-income countries, as well as low- and high-risk bias studies. Heterogeneity of the data was assessed using I2. Cost data was collected for analysis from articles and correspondence with authors. RESULTS: We found a small but significant difference in screening uptake in our primary analysis: RR 1.11 (95% CI: 1.10-1.11; I2 = 97%; 6 trials; 29,018 participants). Our sensitivity analysis, which excluded one trial that measured screening uptake differently than the other trials, resulted in a clearer effect in screening uptake: RR: 1.82 (95% CI: 1.67-1.99; I2 = 42%; 5 trials; 9590 participants). Two trials reported costs; thus, it was not possible to make a direct comparison of costs. One found self-sampling more cost-effective than the provider-required visual inspection with acetic acid method, despite the test and running costs being higher for HPV self-sampling. CONCLUSION: Our review indicates that self-sampling improves screening uptake, particularly in low-income countries; however, to this date, there remain few trials and associated cost data. We recommend further studies with proper cost data be conducted to guide the incorporation of HPV self-sampling into national cervical cancer screening guidelines in low- and middle-income countries. SYSTEMATIC REVIEW REGISTRATION: PROSPERO CRD42020218504.

LINE-1 retrotransposons drive human neuronal transcriptome complexity and functional diversification.

The genetic mechanisms underlying the expansion in size and complexity of the human brain remain poorly understood. Long interspersed nuclear element-1 (L1) retrotransposons are a source of divergent genetic information in hominoid genomes, but their importance in physiological functions and their contribution to human brain evolution are largely unknown. Using multiomics profiling, we here demonstrate that L1 promoters are dynamically active in the developing and the adult human brain. L1s generate hundreds of developmentally regulated and cell type-specific transcripts, many that are co-opted as chimeric transcripts or regulatory RNAs. One L1-derived long noncoding RNA, LINC01876, is a human-specific transcript expressed exclusively during brain development. CRISPR interference silencing of LINC01876 results in reduced size of cerebral organoids and premature differentiation of neural progenitors, implicating L1s in human-specific developmental processes. In summary, our results demonstrate that L1-derived transcripts provide a previously undescribed layer of primate- and human-specific transcriptome complexity that contributes to the functional diversification of the human brain.

CRISPRi-mediated transcriptional silencing in iPSCs for the study of human brain development.

This protocol describes the design and use of CRISPRi-mediated transcriptional silencing in human iPSCs, for loss-of-function studies in brain development research. The protocol avoids single cell selection, thereby eliminating side effects of clonal expansion and sites of viral integration. We also describe a neural progenitor differentiation protocol and discuss the challenges of target-specific lentiviral silencing, efficient silencing levels, and off-target effects. For complete details on the use and execution of this protocol, please refer to Johansson et al. (2022).

SLEEP: intervention with weighted blankets for children with attention deficit hyperactivity disorder (ADHD) and sleep problems: study protocol for a randomised control trial.

INTRODUCTION AND OBJECTIVES: Children with attention deficit hyperactivity disorder (ADHD) have an increased risk of sleep problems. Weighted blankets are one possible non-pharmacological intervention for these problems in this group of children. However, the effectiveness of weighted blankets is insufficiently investigated. This study aims to investigate the effectiveness of weighted blankets in terms of sleep, health-related outcomes and cost-effectiveness as well as to explore children's and parents' experiences of a sleep intervention with weighted blankets. METHODS AND ANALYSIS: This study is a randomised placebo-controlled crossover trial comparing the effect of weighted fibre blankets (active) with fibre blankets without weight (control). Children aged 6-13 years, recently diagnosed with uncomplicated ADHD with verified sleep problems, were included in the study. The study period is 4 weeks for each condition, respectively, and then an 8-week follow-up. A total of 100 children diagnosed with ADHD and sleep problems will enter the study. The primary outcomes are sleep and cost per quality-adjusted life years. The secondary outcomes are health-related quality of life, ADHD symptoms, psychological distress and anxiety. Interviews with a subsample of the participating children and parents will be conducted for exploring the experiences of the intervention. ETHICS AND DISSEMINATION: Ethical approval of the trial has been obtained from the Swedish Ethical Review Authority (number 2019--2158) and conforms to the principles outlined in the Declaration of Helsinki (WMA, 2013). Results will be reported as presentations at peer-review conferences, in articles in peer-review journals and meetings with healthcare providers. TRIAL REGISTRATION NUMBER: NCT04180189.

Empowerment-Based Physical Activity Intervention for People with Advanced Dry Age-Related Macular Degeneration: Mixed-Methods Protocol.

Age-related macular degeneration (AMD) is the most common cause of incurable visual impairment and impacts daily life. These impacts include loss of social activities, decreased functional independence, and reduced physical activity. This protocol aims to describe a prospective, mixed-methodology for studying a population with AMD before, during, and after an empowerment-based physical activity intervention (EPI). A study framework was also developed for EPI. The intervention will include 20 older individuals (age 65+ years) with AMD recruited in Sweden. The intervention period is six months and comprises adapted physical activity and social activities in a group twice a week and individual health coaching on three occasions. The quantitative pre-test and three follow-ups include physical functional tests, an accelerometer that monitors physical activity continuously for one week, and questionnaires. Individual and focus-group interviews and ethnographic observations will explore the experience of living with AMD and what it means to participate in the EPI for individuals with AMD. The chosen methodology offers a structured way for researchers to explore the experiences and factors that may provide insights into the potential of creative supervised, adapted physical activity in groups, health coaching, and socialising that are significant to enable well-being among older individuals with AMD.

A cis-acting structural variation at the ZNF558 locus controls a gene regulatory network in human brain development.

The human forebrain has expanded in size and complexity compared to chimpanzees despite limited changes in protein-coding genes, suggesting that gene expression regulation is an important driver of brain evolution. Here, we identify a KRAB-ZFP transcription factor, ZNF558, that is expressed in human but not chimpanzee forebrain neural progenitor cells. ZNF558 evolved as a suppressor of LINE-1 transposons but has been co-opted to regulate a single target, the mitophagy gene SPATA18. ZNF558 plays a role in mitochondrial homeostasis, and loss-of-function experiments in cerebral organoids suggests that ZNF558 influences developmental timing during early human brain development. Expression of ZNF558 is controlled by the size of a variable number tandem repeat that is longer in chimpanzees compared to humans, and variable in the human population. Thus, this work provides mechanistic insight into how a cis-acting structural variation establishes a regulatory network that affects human brain evolution.

Financing intersectoral health promotion programmes: some reasons why collaborators are collaborating as indicated by cost-effectiveness analyses.

AIMS: Intersectoral collaboration is an important part of many health promotion programmes. The reasons for the local organisations to collaborate, i.e. to finance programmes, are presumably based on benefits they derive from the collaboration. The aim of this study is to discuss whether subsector financial analyses based on data from cost-effectiveness analyses reflect incentives of collaborating organisations in two intersectoral health promotion programmes. METHODS: Within economics, financial incentives are important reasons for actions. The financial incentives of collaborators are exemplified with two subsector financial analyses containing avoided disease-related costs as estimated in two cost-effectiveness analyses, on an elderly safety promotion programme (Safe Seniors in Sundbyberg) and on a diabetes prevention programme (Stockholm Diabetes Prevention Program, SDPP) from Stockholm, Sweden. RESULTS: The subsector financial analyses indicate that there are financial incentives for the key local community organisation, i.e. the local authority, to collaborate in one of the programmes but not the other. There are no financial benefits for other important community organisations, such as non-governmental organisations. CONCLUSIONS: The reasons for collaborating organisations to collaborate within intersectoral health promotion programmes extend beyond financial benefits from averted disease. Thus, the reported subsector financial analyses are only partial reflections of the incentives of collaborators, but they might be used as a starting point for discussions on cost sharing among potential intersectoral collaborators.

Patients' opinion on the use of 2 generations of power-driven water flossers and their impact on gingival inflammation.

OBJECTIVES: To assess patients' opinion on the use of 2 generations of power-driven water flossers and their impact on gingival inflammation. MATERIAL & METHODS: In the present prospective cohort study 24 periodontitis patients under regular supportive periodontal therapy used daily 2 generations of a power-driven water flosser (Sonicare AirFloss [SAF] and Sonicare AirFloss Ultra [SAFU]) for 12 weeks each. Patients were instructed to position the nozzle interproximally from the buccal aspect at each interproximal space. Patients' opinion was assessed by a questionnaire and interproximal bleeding on probing (BoP) was recorded. RESULTS: Overall satisfaction with SAF/SAFU was rated high, by about 80% of the patients. About 66% of the patients preferred SAF/SAFU compared to their previous interdental cleaning device and indicated that they would continue using SAF/SAFU after the study; none of the patients reported any discomfort or pain. Compared to only tooth brushing, daily use of SAF/SAFU caused a significant reduction of interproximal BoP values, which were well maintained over 6 months; that is, BoP at interproximal buccal and oral sites (pooled), as well as at interproximal buccal and oral sites separately, was proportionately reduced by 29.1%, 41.2%, and 24.8%, respectively (pooled: p = 0.027; buccal sites: p = 0.030; oral sites: p = 0.030). CONCLUSION: Patients were very fond of the power-driven water flossers tested herein, and daily use of the devices for 6 months (i.e., each device was used for 3 months) resulted in a significant reduction of gingival inflammation interproximally.

Bacterial colonization of a power-driven water flosser during regular use. A proof-of-principle study.

OBJECTIVES: The present proof-of-principle study assessed whether daily use of a power-driven water flosser (Sonicare AirFloss; SAF) leads to bacterial colonization in the nozzle and/or the device, resulting in contaminated water-jet. MATERIAL AND METHODS: In five participants, saliva samples at baseline and water-jet samples of devices used daily with bottled water for 3 weeks (test) were collected. Additionally, water-jet samples from devices used daily with bottled water extra-orally for 3 weeks (positive control) and from brand new devices (negative control), as well as samples from newly opened and 1- and 3-week opened water bottles were collected. Colony forming units (CFU) were recorded after 48 h culturing and 20 oral pathogens were assessed by polymerase chain reaction-based analysis. RESULTS: Distinct inter-individual differences regarding the number of detected bacteria were observed; water-jet samples of test devices included both aerobic and anaerobic bacterial species, with some similarities to the saliva sample of the user. Water-jet samples from positive control devices showed limited number of aerobic and anaerobic bacterial species, while the samples from negative control devices did not show any bacterial species. Very few aerobic bacteria were detected only in the 3-week-old bottled water samples, while samples of newly and 1-week opened water bottles did not show any bacterial growth. CONCLUSIONS: The present proof-of-principle study showed that daily use of a power-driven water flosser for 3 weeks resulted in bacterial colonization in the nozzle and/or device with both aerobic and anaerobic, not only oral, species, that are transmitted via the water-jet.