The Genetic Basis of Probable REM Sleep Behavior Disorder in Parkinson's Disease.

Patients with Parkinson's Disease (PD) experience REM sleep behavior disorder (RBD) more frequently than healthy controls. RBD is associated with torpid disease evolution. To test the hypothesis that differential genetic signatures might contribute to the torpid disease evolution in PD patients with RBD we compared the rate of genetic mutations in PD patients with or without probable RBD. Patients with a clinical diagnosis of PD in the Parkinson's Progression Markers Initiative (PPMI) database entered the study. We excluded those with missing data, dementia, psychiatric conditions, or a diagnosis change over the first five years from the initial PD diagnosis. Probable RBD (pRBD) was confirmed by a REM Sleep Behavior Disorder Screening Questionnaire score > 5 points. Logistic regression and Machine Learning (ML) algorithms were used to relate Single Nucleotide Polymorphism (SNPs) in PD-related genes with pRBD. We included 330 PD patients fulfilling all inclusion and exclusion criteria. The final logistic multivariate model revealed that the following SNPs increased the risk of pRBD: GBA_N370S_rs76763715 (OR, 95% CI: 3.38, 1.45-7.93), SNCA_A53T_rs104893877 (8.21, 2.26-36.34), ANK2. CAMK2D_rs78738012 (2.12, 1.08-4.10), and ZNF184_rs9468199 (1.89, 1.08-3.33). Conversely, SNP COQ7. SYT17_rs11343 reduced pRBD risk (0.36, 0.15-0.78). The ML algorithms led to similar results. The predictive models were highly specific (95-99%) but lacked sensitivity (9-39%). We found a distinctive genetic signature for pRBD in PD. The high specificity and low sensitivity of the predictive models suggest that genetic mutations are necessary but not sufficient to develop pRBD in PD. Additional investigations are needed.

Neuroprotection from protein misfolding in cerebral hypoperfusion concurrent with metabolic syndrome. A translational perspective.

Based on clinical and experimental evidence, metabolic syndrome (MetS) and type 2 diabetes (T2D) are considered risk factors for chronic cerebral hypoperfusion (CCH) and neurodegeneration. Scientific evidence suggests that protein misfolding is a potential mechanism that explains how CCH can lead to either Alzheimer's disease (AD) or vascular cognitive impairment and dementia (VCID). Over the last decade, there has been a significant increase in the number of experimental studies regarding this issue. Using several animal paradigms and different markers of CCH, scientists have discussed the extent to which MetSor T2D causes a decrease in cerebral blood flow (CBF). In addition, different models of CCH have explored how long-term reductions in oxygen and energy supply can trigger AD or VCID via protein misfolding and aggregation. Research that combines two or three animal models could broaden knowledge of the links between these pathological conditions. Recent experimental studies suggest novel neuroprotective properties of protein-remodeling factors. In this review, we present a summarized updated revision of preclinical findings, discussing clinical implications and proposing new experimental approaches from a translational perspective. We are confident that research studies, both clinical and experimental, may find new diagnostic and therapeutic tools to prevent neurodegeneration associated with MetS, diabetes, and any other chronic non-communicable disease (NCD) associated with diet and lifestyle risk factors.

Development and Validation of the Dystonia-Pain Classification System: A Multicenter Study.

BACKGROUND: Dystonia is associated with disabling nonmotor symptoms like chronic pain (CP), which is prevalent in dystonia and significantly impacts the quality of life (QoL). There is no validated tool for assessing CP in dystonia, which substantially hampers pain management. OBJECTIVE: The aim was to develop a CP classification and scoring system for dystonia. METHODS: A multidisciplinary group was established to develop the Dystonia-Pain Classification System (Dystonia-PCS). The classification of CP as related or unrelated to dystonia was followed by the assessment of pain severity score, encompassing pain intensity, frequency, and impact on daily living. Then, consecutive patients with inherited/idiopathic dystonia of different spatial distribution were recruited in a cross-sectional multicenter validation study. Dystonia-PCS was compared to validated pain, mood, QoL, and dystonia scales (Brief Pain Inventory, Douleur Neuropathique-4 questionnaire, European QoL-5 Dimensions-3 Level Version, and Burke-Fahn-Marsden Dystonia Rating Scale). RESULTS: CP was present in 81 of 123 recruited patients, being directly related to dystonia in 82.7%, aggravated by dystonia in 8.8%, and nonrelated to dystonia in 7.5%. Dystonia-PCS had excellent intra-rater (Intraclass Correlation Coefficient - ICC: 0.941) and inter-rater (ICC: 0.867) reliability. In addition, pain severity score correlated with European QoL-5 Dimensions-3 Level Version's pain subscore (r = 0.635, P < 0.001) and the Brief Pain Inventory's severity and interference scores (r = 0.553, P < 0.001 and r = 0.609, P < 0.001, respectively). CONCLUSIONS: Dystonia-PCS is a reliable tool to categorize and quantify CP impact in dystonia and will help improve clinical trial design and management of CP in patients affected by this disorder. © 2023 The Authors. Movement Disorders published by Wiley Periodicals LLC on behalf of International Parkinson and Movement Disorder Society.

Genetics of Neurogenic Orthostatic Hypotension in Parkinson's Disease, Results from a Cross-Sectional In Silico Study.

The genetic basis of Neurogenic Orthostatic Hypotension (NOH) in Parkinson's disease (PD) has been inadequately explored. In a cross-sectional study, we examined the association between NOH and PD-related single-nucleotide polymorphisms (SNPs) and mapped their effects on gene expression and metabolic and signaling pathways. Patients with PD, free from pathological conditions associated with OH, and not taking OH-associated medications were included. NOH was defined as per international guidelines. Logistic regression was used to relate SNPs to NOH. Linkage-disequilibrium analysis, expression quantitative trait loci, and enrichment analysis were used to assess the effects on gene expression and metabolic/signaling pathways. We included 304 PD patients in the study, 35 of whom had NOH (11.5%). NOH was more frequent in patients with SNPs in SNCA, TMEM175, FAM47E-STBD1, CCDC62, SCN3A, MIR4696, SH3GL2, and LZTS3/DDRGK1 and less frequent in those with SNPs in ITGA8, IP6K2, SIPA1L2, NDUFAF2. These SNPs affected gene expression associated with the significant hierarchical central structures of the autonomic nervous system. They influenced several metabolic/signaling pathways, most notably IP3/Ca++ signaling, the PKA-CREB pathway, and the metabolism of fatty acids. These findings provide new insights into the pathophysiology of NOH in PD and may provide targets for future therapies.

Screen use, sleep duration, daytime somnolence, and academic failure in school-aged adolescents.

In this study, we examined the relationship between screen time use, sleep characteristics, daytime somnolence, and academic performance in school-aged adolescents. We surveyed 1,257 12- to 18-year-old adolescents attending 52 schools in urban or suburban areas of Argentina. We recorded the daily exposure to various screen-based activities, including video- and online-gaming, social media, TV or streaming. Screen time and device type in the hour before bedtime, sleep patterns during weekdays and weekends, somnolence (Pediatric Daytime Sleepiness Scale score), and grades in language and mathematics were also assessed. Structural Equation Modelling was used to identify a path connecting the latent variables. Results are expressed as standardized regression weights (srw). Missing data were present in 393 subjects, and thus the final sample consisted of 864 complete responses. Daytime somnolence (i.e., PDSS score ≥ 15) was observed in 614 participants (71%), and academic failure (i.e., grades < 7/10) in 352 of them (41%). Time spent using video gaming consoles was negatively associated with sleep duration (srw = -0.22, p<0.01) and positively connected with daytime somnolence (srw = 0.11, p<0.01). Use of mobile devices was associated with lower academic performance (srw = -0.11, p<0.01). Sleep duration was inversely related to daytime somnolence (srw = -0.27, p<0.01), which was in turn negatively associated with academic performance (srw = -0.18, p<0.05). Bedtime computer use did not influence any outcome. In summary, among adolescents, screen use adversely affected nighttime sleep, daytime somnolence, and academic performance. These findings call for the implementation of educational public campaigns aimed at promoting healthy sleep and reducing screen exposure among adolescents.

A systematic review and meta-analysis on the association between orthostatic hypotension and mild cognitive impairment and dementia in Parkinson's disease.

BACKGROUND: Cognitive impairment is a frequent disabling feature of Parkinson's disease (PD). Orthostatic hypotension (OH) is treatable and may be a risk factor for cognitive impairment. OBJECTIVE: We conducted a systematic review and meta-analysis to examine the relationship between OH with PD-associated minimal cognitive impairment (PD-MCI) and dementia (PDD) and assess the mitigating effects of potential confounding factors. METHODS: Observational studies published in English, Spanish, French, or Portuguese up to January 2022 were searched for in PubMed, EBSCO, and SciELO databases. The primary aim of this study was to revise the association between OH with PD-MCI and PDD. Alongside, we assessed OH as related to cognitive rating scales. Fixed and random models were fitted. Meta-regression was used to assess the mitigating effects of confounding variables. RESULTS: We identified 18 studies that reported OH association with PDD or PD-MCI, 15 of them reporting OH association with cognitive rating scales. OH was significantly associated with PDD/PD-MCI (OR, 95% CI: 3.31, 2.16-5.08; k = 18, n = 2251; p < 0.01). OH association with PDD (4.64, 2.68-8.02; k = 13, n = 1194; p < 0.01) was stronger than with PD-MCI (1.82, 0.92-3.58; k = 5, n = 1056; p = NS). The association between OH and PD-MCI/PDD was stronger in studies with a higher proportion of women and in those with a lower frequency of supine hypertension. Global cognition rating scale scores were lower in patients with OH (SMD, 95% CI: - 0.55, - 0.83/ - 0.26; k = 12, n = 1427; p < 0.01). CONCLUSIONS: Orthostatic hypotension shows as a significant risk factor for cognitive impairment in PD, especially in women and patients not suffering from hypertension.

Disproportionality analysis of adverse neurological and psychiatric reactions with the ChAdOx1 (Oxford-AstraZeneca) and BNT162b2 (Pfizer-BioNTech) COVID-19 vaccines in the United Kingdom.

BACKGROUND: Information on neurological and psychiatric adverse events following immunization (AEFIs) with COVID-19 vaccines is limited. RESEARCH DESIGN & METHODS: We examined and compared neurological and psychiatric AEFIS reports related to BNT162b2 (Pfizer-BioNTech) and ChAdOx1 (Oxford-AstraZeneca) COVID-19 vaccines and recorded in the United Kingdom Medicines and Healthcare products Regulatory Agency between 9 December 2020 and 30 June 2021. RESULTS: As of 30 June 2021, 46.1 million doses of ChAdOx1 and 30.3 million doses of BNT162b2 had been administered. The most frequently reported AEFI was headache with 1,686 and 575 cases per million doses of ChAdOx1 and BNT162b2, respectively. AEFIs more frequently reported after CHAdOx1 compared with BNT162b2 vaccination were Guillain-Barré syndrome (OR, 95% CI = 2.53, 1.82-3.51), freezing (6.66, 3.12-14.22), cluster headache (1.53, 1.28-1.84), migraine (1.23,1.17-1.30), postural dizziness (1.24,1.13-1.37), tremor (2.86, 2.68-3.05), headache (1.40, 1.38-1.43), paresthesia (1.11, 1.06-1.16), delirium (1.85, 1.45-2.36), hallucination (2.20, 1.82-2.66), poor quality sleep (1.53, 1.26-1.85), and nervousness (1.54, 1.26-1.89) Reactions less frequently reported with ChAdOx1 than with BNT162b2 were Bell's palsy (0.47, 0.41-0.55), anosmia (0.58, 0.47-0.71), facial paralysis (0.35, 0.29-0.41), dysgeusia (0.68, 0.62-0.73), presyncope (0.48, 0.42-0.55), syncope (0.63, 0.58-0.67), and anxiety (0.75 (0.67-0.85). CONCLUSION: Neurological and psychiatric AEFIs were relatively infrequent, but each vaccine was associated with a distinctive toxic profile.

We examined reports on adverse neurological and psychiatric effects following immunization with BNT162b2 (Pfizer-BioNTech) and ChAdOx1 (Oxford-AstraZeneca) for COVID-19 to the United Kingdom Medicines and Healthcare products Regulatory Agency between 9 December 2020 and 30 June 2021. Adverse effects following immunization (AEFIs) were relatively infrequent. Compared to BNT162b2, Guillain-Barré syndrome, freezing phenomenon, cluster headache, migraine, postural dizziness, tremor, headache, paresthesia, delirium, hallucination, poor quality sleep, and nervousness were more frequently reported for ChAdOx1. Reactions less frequently reported for ChAdOx1 than for BNT162b2 were Bell's palsy, anosmia, facial paralysis, dysgeusia, presyncope, syncope, and anxiety.

Melatonin as a Chronobiotic and Cytoprotective Agent in Parkinson's Disease.

This article discusses the role that melatonin may have in the prevention and treatment of Parkinson's disease (PD). In parkinsonian patients circulating melatonin levels are consistently disrupted and the potential therapeutic value of melatonin on sleep disorders in PD was examined in a limited number of clinical studies using 2-5 mg/day melatonin at bedtime. The low levels of melatonin MT1 and MT2 receptor density in substantia nigra and amygdala found in PD patients supported the hypothesis that the altered sleep/wake cycle seen in PD could be due to a disrupted melatonergic system. Motor symptomatology is seen in PD patients when about 75% of the dopaminergic cells in the substantia nigra pars compacta region degenerate. Nevertheless, symptoms like rapid eye movement (REM) sleep behavior disorder (RBD), hyposmia or depression may precede the onset of motor symptoms in PD for years and are index of worse prognosis. Indeed, RBD patients may evolve to an α-synucleinopathy within 10 years of RBD onset. Daily bedtime administration of 3-12 mg of melatonin has been demonstrated effective in RDB treatment and may halt neurodegeneration to PD. In studies on animal models of PD melatonin was effective to curtail symptomatology in doses that allometrically projected to humans were in the 40-100 mg/day range, rarely employed clinically. Therefore, double-blind, placebo-controlled clinical studies are urgently needed in this respect.

The Renin-Angiotensin System Modulates Dopaminergic Neurotransmission: A New Player on the Scene.

Parkinson's disease (PD) is an extrapyramidal disorder characterized by neuronal degeneration in several regions of the peripheral and central nervous systems. It is the second most frequent neurodegenerative disease after Alzheimer's. It has become a major health problem, affecting 1% of the world population over 60 years old and 3% of people beyond 80 years. The main histological findings are intracellular Lewy bodies composed of misfolded α-synuclein protein aggregates and loss of dopaminergic neurons in the central nervous system. Neuroinflammation, apoptosis, mitochondrial dysfunction, altered calcium homeostasis, abnormal protein degradation, and synaptic pathobiology have been put forward as mechanisms leading to cell death, α-synuclein deposition, or both. A progressive loss of dopaminergic neurons in the substantia nigra late in the neurodegeneration leads to developing motor symptoms like bradykinesia, tremor, and rigidity. The renin-angiotensin system (RAS), which is involved in regulating blood pressure and body fluid balance, also plays other important functions in the brain. The RAS is involved in the autocrine and paracrine regulation of the nigrostriatal dopaminergic synapses. Dopamine depletion, as in PD, increases angiotensin II expression, which stimulates or inhibits dopamine synthesis and is released via AT1 or AT2 receptors. Furthermore, angiotensin II AT1 receptors inhibit D1 receptor activation allosterically. Therefore, the RAS may have an important modulating role in the flow of information from the brain cortex to the basal ganglia. High angiotensin II levels might even aggravate neurodegeneration, activating the nicotinamide adenine dinucleotide phosphate (NADPH) oxidase complex, which leads to increased reactive oxygen species production.

Effects of angiotensin type 1 receptor antagonists on Parkinson's disease progression: An exploratory study in the PPMI database.

INTRODUCTION: We explored the potential clinical effects of angiotensin-II AT1 receptor blockers (ARBs) and angiotensin-converting enzyme inhibitors (ACEIs) in patients from the Parkinson's Progress Marker Initiative (PPMI) study database. METHODS: We included 423 newly diagnosed PD patients, free from antiparkinsonian treatment, from the PPMI. We compared the proportion of patients starting on l-DOPA during the first year of follow-up, and the changes in MDS-UPDRS total score and sub-scores during the first five follow-up years for patients exposed or not to ARBs or ACEIs. RESULTS: Treatment with ARBs did not affect the proportion of patients on l-DOPA during the first year (adjusted OR, 95% CI = 0.26, 0.03-2.18, N.S.) while reduced MDS-UPDRS total score (0.85, 0.76-0.95, p < 0.01). Patients treated with ACEIs experienced no changes in either measure. CONCLUSIONS: These results show potential signals for a beneficial effect with ARBs. Further clinical trials are warranted.

Assessment of Ataxia Rating Scales and Cerebellar Functional Tests: Critique and Recommendations.

BACKGROUND: We assessed the clinimetric properties of ataxia rating scales and functional tests, and made recommendations regarding their use. METHODS: A systematic literature search was conducted to identify the instruments used to rate ataxia symptoms. The identified rating scales and functional ability tests were reviewed and ranked by the panel as "recommended," "suggested," or "listed" for the assessment of patients with discrete cerebellar disorders, using previously established criteria. RESULTS: We reviewed 14 instruments (9 rating scales and 5 functional tests). "Recommended" rating scales for the assessment of symptoms severity were: for Friedreich's ataxia, the Friedreich's Ataxia Rating Scale, the International Cooperative Ataxia Rating Scale (ICARS), and the Scale for the Assessment and Rating of Ataxia (SARA); for spinocerebellar ataxias, ICARS and SARA; for ataxia telangiectasia: ICARS and SARA; for brain tumors, SARA; for congenital disorder of glycosylation-phosphomannomutase-2 deficiency, ICARS; for cerebellar symptoms in multiple sclerosis, ICARS; for cerebellar symptoms in multiple system atrophy: Unified Multiple System Atrophy Rating Scale and ICARS; and for fragile X-associated tremor ataxia syndrome, ICARS. "Recommended" functional tests were: for Friedreich's ataxia, Ataxia Functional Composite Score and Composite Cerebellar Functional Severity Score; and for spinocerebellar ataxias, Ataxia Functional Composite Score, Composite Cerebellar Functional Severity Score, and SCA Functional Index. CONCLUSIONS: We identified some "recommended" scales and functional tests for the assessment of patients with major hereditary ataxias and other cerebellar disorders. The main limitations of these instruments include the limited assessment of patients in the more severe end of the spectrum and children. Further research in these populations is warranted. © 2020 International Parkinson and Movement Disorder Society.

Sleep disruption in medicine students and its relationship with impaired academic performance: A systematic review and meta-analysis.

Sleep disruption severely impairs learning ability, affecting academic performance in students. This systematic review and meta-analysis aimed at assessing the prevalence of sleep disruption in medical students and its relationship with academic performance. PubMed, Web of Sciences, EBSCO and SciELO databases searches allowed to retrieve 41 papers with data about the prevalence of sleep deprivation, 20 of which also contained data on its association with academic performance. Poor sleep quality was reported by 5646 out of 14,170 students in 29 studies (39.8%, 95% confidence interval = 39.0-40.6%), insufficient sleep duration by 3762/12,906 students in 28 studies (29.1%, 23.3-29.9%) and excessive diurnal sleepiness by 1324/3688 students in 13 studies (35.9%, 34.3-37.4). Academic grades correlated significantly with sleep quality scores (r, 95% CI = 0.15, 0.05-0.26, random-effects model; p = 0.002, n = 10,420 subjects, k = 15 studies) and diurnal sleepiness (r = -0.12, -0.19/-0.06 under the fixed effects model, p < 0.001, n = 1539, k = 6), but not with sleep duration (r = 0.03, -0.12/0.17 under the random-effects model, p = 0.132, n = 2469, k = 9). These findings advocate for an urgent intervention aiming at improving sleep quality among medical students as a way of increasing academic achievements and, ultimately, the quality of health care.

Role of Astrocytic Dysfunction in the Pathogenesis of Parkinson's Disease Animal Models from a Molecular Signaling Perspective.

Despite the fact that astrocytes are the most abundant glial cells, critical for brain function, few studies have dealt with their possible role in neurodegenerative diseases like Parkinson's disease (PD). This article explores relevant evidence on the involvement of astrocytes in experimental PD neurodegeneration from a molecular signaling perspective. For a long time, astrocytic proliferation was merely considered a byproduct of neuroinflammation, but by the time being, it is clear that astrocytic dysfunction plays a far more important role in PD pathophysiology. Indeed, ongoing experimental evidence suggests the importance of astrocytes and dopaminergic neurons' cross-linking signaling pathways. The Wnt-1 (wingless-type MMTV integration site family, member 1) pathway regulates several processes including neuron survival, synapse plasticity, and neurogenesis. In PD animal models, Frizzled (Fzd) neuronal receptors' activation by the Wnt-1 normally released by astrocytes following injuries leads to ß-catenin-dependent gene expression, favoring neuron survival and viability. The transient receptor potential vanilloid 1 (TRPV1) capsaicin receptor also participates in experimental PD genesis. Activation of astrocyte TRPV1 receptors by noxious stimuli results in reduced inflammatory response and increased ciliary neurotrophic factor (CNTF) synthesis, which enhances neuronal survival and differentiation. Another major pathway involves IκB kinase (IKK) downregulation by ARL6ip5 (ADP-ribosylation-like factor 6 interacting protein 5, encoded by the cell differentiation-associated, JWA, gene). Typically, IKK releases the proinflammatory NF-κB (nuclear factor kappa-light-chain-enhancer of activated B cells) molecule from its inhibitor. Therefore, by downregulating NF-κB inhibitor, ARL6ip5 promotes an anti-inflammatory response. The evidence provided by neurotoxin-induced PD animal models guarantees further research on the neuroprotective potential of normalizing astrocyte function in PD.

State-of-the-art pharmacotherapy for autonomic dysfunction in Parkinson's disease.

Introduction: Autonomic dysfunction is one of the most frequent and disabling non-motor symptoms of Parkinson's disease (PD). It includes, among others, orthostatic hypotension (OH), sialorrhea, constipation, erectile dysfunction (ED), urinary dysfunction, and diaphoresis. They are usually under-recognized and suboptimally managed.Areas covered: Recommended treatments for dysautonomias are summarized with a description of the mechanism of action and observed results. The pathophysiology of each disorder is reviewed to pinpoint possible therapeutic targets. Drugs approved for treating dysautonomia in the general population along with those under development for PD-related dysautonomia are also reviewed. Finally, the key elements of each symptom that should be addressed in clinical trials' design are considered.Expert opinion: Midodrine, droxidopa, fludrocortisone, and domperidone may be used for OH treatment. Sialorrhea can be managed with botulin toxin injections and oral glycopyrrolate. Erectile dysfunction can benefit from sildenafil treatment, as urinary dysfunction can from solifenacin. Macrogol, lubiprostone, and probiotics might be effective in treating constipation. Further research is needed to determine adequate treatment for diaphoresis in PD patients. Multidisciplinary management of motor and non-motor symptoms in PD is the best approach for dysautonomias in PD.

Prevalence and factors related to orthostatic syndromes in recently diagnosed, drug-naïve patients with Parkinson disease.

PURPOSE: The aim of this study was to explore the prevalence of and factors related to orthostatic syndromes in recently diagnosed drug-naïve patients with Parkinson disease (PD). METHODS: This was a cross-sectional study that included 217 drug-naïve patients with PD and 108 sex- and age-matched non-parkinsonian controls from the Parkinson's Progression Markers Initiative (PPMI) prospective cohort study who were devoid of diabetes, alcoholism, polyneuropathy, amyloidosis, and hypotension-inducing drugs. Orthostatic symptoms were evaluated using the Scales for Outcomes in PD-Autonomic Dysfunction (SCOPA-AUT). Ioflupane-I123 single-photon emission computerized tomography was used to evaluate striatal dopamine active transporter (DaT) levels. Blood pressure was assessed both in the supine position and 1-3 min after the switch to a standing position. Orthostatic hypotension (OH) was defined by international consensus, and orthostatic intolerance (OI) was defined as the presence of orthostatic symptoms in the absence of OH. RESULTS: Compared with non-parkinsonian controls, patients with PD experienced a mild fall in systolic blood pressure upon standing (p = 0.082). The prevalence of OH was 11.1% in PD patients and 5.6% in controls (p = 0.109). The prevalence of OI was higher in patients with PD than in controls (31.3 vs. 13.3%; p = 0.003). Logistic regression revealed that OH and OI were related to a lower striatal DaT level and higher SCOPA-AUT gastrointestinal score. CONCLUSIONS: Orthostatic syndromes were common in the recently diagnosed drug-naïve patients with PD enrolled in the study, but only the prevalence of OI was higher in PD patients than in the non-parkinsonian controls. Unlike motor or functional disability indicators, markers of dopaminergic striatal deficit and gastrointestinal dysfunction were associated with OH and OI.