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The unicellular green alga Chlamydomonas reinhardtii adapts to anaerobic or hypoxic conditions by developing a complex fermentative metabolism including the production of molecular hydrogen by [FeFe]-hydrogenase isoform1 (HYDA1). HYDA1 transcript and hydrogenase protein accumulate in the absence of oxygen or copper (Cu). Factors regulating this differential gene expression have been unknown so far. In this study, we report on the isolation of a Chlamydomonas mutant strain impaired in HYDA1 gene expression by screening an insertional mutagenesis library for HYDA1 promoter activity using the arylsulfatase-encoding ARYLSULFATASE2 gene as a selection marker. The mutant strain has a deletion of the COPPER RESPONSE REGULATOR1 (CRR1) gene encoding for CRR1, indicating that this SQUAMOSA-PROMOTER BINDING PROTEIN (SBP) domain transcription factor is involved in the regulation of HYDA1 transcription. Treating the C. reinhardtii wild type with mercuric ions, which were shown to inhibit the binding of the SBP domain to DNA, prevented or deactivated HYDA1 gene expression. Reporter gene analyses of the HYDA1 promoter revealed that two GTAC motifs, which are known to be the cores of CRR1 binding sites, are necessary for full promoter activity in hypoxic conditions or upon Cu starvation. However, mutations of the GTAC sites had a much stronger impact on reporter gene expression in Cu-deficient cells. Electrophoretic mobility shift assays showed that the CRR1 SBP domain binds to one of the GTAC cores in vitro. These combined results prove that CRR1 is involved in HYDA1 promoter activation.

IntroductionIn May 2019, the WHO classified internet gaming disorder (IGD) as a mental disorder in the upcoming International Classification of Diseases 11th Revision. However, individuals affected by IGD or internet use disorders (IUDs) are often not provided with adequate therapy due to a lack of motivation or absence of adequate local treatment options. To close the gap between individuals with IUDs and the care system, we conduct an online-based motivational intervention to reduce problematic internet use and promote treatment motivation in internet gaming disorder and internet use disorder (OMPRIS).Methods and analysisWithin the randomised controlled trial, a total of n=162 participants will be allocated by sequential balancing randomisation to the OMPRIS intervention or a waitlist control group. The study includes an extensive diagnostic, followed by a 4-week psychological intervention based on motivational interviewing, (internet-related) addiction therapy, behavioural therapy techniques and additional social counselling. The primary outcome is the reduction of problematic internet use measured by the Assessment of Internet and Computer Game Addiction Scale. Secondary outcomes include time spent on the internet, motivation for change (Stages of Change Readiness and Treatment Eagerness Scale for Internet Use Disorder), comorbid mental symptoms (Patient Health Questionnaire-9, Generalized Anxiety Disorder Screener-7), quality of life (EuroQoL Standardised Measure of Health-related Quality of Life–5 Dimensions, General Life Satisfaction-1), self-efficacy (General Self-Efficacy Scale), personality traits (Big Five Inventory-10), therapeutic alliance (Helping Alliance Questionnaire) and health economic costs. The diagnosis of (comorbid) mental disorders is carried out with standardised clinical interviews. The measurement will be assessed before (T0), at midpoint (T1) and after the OMPRIS intervention (T2), representing the primary endpoint. Two follow-up assessments will be conducted after 6 weeks (T3) and 6 months (T4) after the intervention. The outcomes will be analysed primarily via analysis of covariance. Both intention-to-treat and per-protocol analyses will be conducted.Ethics and disseminationParticipants will provide written informed consent. The trial has been approved by the Ethics Committee of the Faculty of Medicine, Ruhr University Bochum (approval number 19-6779). Findings will be disseminated through presentations, peer-reviewed journals and conferences.Trial registration numberDRKS00019925.