NPC1 allelic heterogeneity. Importantly, 73 P/LP variants were previously unpublished. The most frequent variants detected were: c.3019C > G, p.(P1007A), c.3104C > T, p.(A1035V), and c.2861C > T, p.(S954L). Loss of function (LoF) variants were significantly associated with earlier age at diagnosis, highly increased biomarker levels, and a visceral phenotype . On the other hand, the variants p.(P1007A) and p.(S954L) were significantly associated with later age at diagnosis (p < 0.001) and mildly elevated biomarker levels (p ≤ 0.002), consistent with the juvenile/adult form of NPC1. In addition, p.(I1061T), p.(S954L), and p.(A1035V) were associated with abnormality of eye movements . We describe the largest and most heterogenous cohort of NPC1 patients published to date. Our results suggest that besides its utility in variant classification, the biomarker PPCS might serve to indicate disease severity/progression. In addition, we establish new genotype–phenotype relationships for “frequent” NPC1 variants.Niemann-Pick type C1 disease (NPC1 [OMIM 257220]) is a rare and severe autosomal recessive disorder, characterized by a multitude of neurovisceral clinical manifestations and a fatal outcome with no effective treatment to date. Aiming to gain insights into the genetic aspects of the disease, clinical, genetic, and biomarker PPCS data from 602 patients referred from 47 countries and diagnosed with NPC1 in our laboratory were analyzed. Patients’ clinical data were dissected using Human Phenotype Ontology (HPO) terms, and genotype–phenotype analysis was performed. The median age at diagnosis was 10.6 years (range 0–64.5 years), with 287 unique pathogenic/likely pathogenic (P/LP) variants identified, expanding  Niemann-Pick type C1 disease (NPC1 OMIM 257220]) is a rare genetic disorder presenting numerous neurovisceral clinical manifestations. Although the pathophysiology of NPC is complex, the most well-accepted hypothesis postulates that an intracellular disruption of the cholesterol transport causes accumulation of unesterified cholesterol and other molecules in the late endosomal/lysosomal compartments. Consequently, the abnormal accumulation of these substances leads to damage and degeneration of various cells and tissues of the body, in particular the neurons in the central nervous system [57220) isThe clinical presentation of NPC1 is heterogeneous and usually includes neurodevelopmental delay, cognitive impairment, ataxia, abnormal ocular movements, psychosis, mood disorders, dementia, and hepatosplenomegaly. In addition, the age of onset of clinical symptoms is highly variable and can range between the neonatal period through adulthood. Furthermore, age of onset of neurological symptoms directly correlates to life expectancy and could be used to predict disease severity , 4. The NPC1 gene [NPC1 gene are accountable for the disease [NPC2 gene [NPC1 is inherited as an autosomal recessive disease and is caused by the biallelic pathogenic or likely pathogenic (P/LP) variants in the PC1 gene , 9. To d disease . A similPC2 gene , 11. NPCPC2 gene .NPC1/NPC2 analysis, or dedicated gene panels, which can be combined with enzymatic assay testing of acidic sphingomyelinase and the biomarker N-palmitoyl-O-phosphocholineserine  [Establishing the diagnosis of NPC and prediction of disease course is challenging given the heterogeneous clinical presentations, variable age of onset, and allelic heterogeneity of the disease . Genetic-SM-509) , 15.NPC1, including the report of 73 novel causative variants, and their geographical aggregation. Furthermore, we disclose novel genotype-HPO terms-biomarker relationships and illustrate the value of combined genetic/biomarker testing to diagnose and increase the understanding of NPC1.With this work, we describe the largest cohort of genetically diagnosed NPC1 patients reported to date. The analysis of our Biodatabank included clinical, genetic, and biomarker results from this group of 602 NPC1 patients. Our results expand the allelic heterogeneity of The current project has been conducted within a diagnostic setting, and in the second step, utilized deidentified data and samples—thus not requiring Institutional Review Boards (IRB) approval.NPC1 variants between October 2006 and March 2021. Information was compiled related to age at diagnosis, gender, and country of origin along with available clinical, genetic, and biomarker data. The clinical information provided by referring clinicians was converted into HPO terms by a dedicated team of scientists. During this process, the affection status of the individual, age at onset, and family history, as well as the curated HPO terms were registered in our laboratory management system (LIMS) and the CENTOGENE Biodatabank. Every provided document was carefully inspected, and clinical terms were marked and then stored for quality control purposes. In cases of contradictory or unclear information, the referring clinician was contacted for clarification.We queried the CENTOGENE Biodatabank for unrelated (consecutive) patients who received a genetic diagnosis of NPC1 based on the detection of biallelic P/LP ®) (Table NPC1 (NM_000271.4) were available on request. MLPA® analyses were performed with commercially available kits according to manufacturer’s instructions . MLPA reactions were run in ABI 3730xl/3130xl DNA Analyzers (Applied Biosystems).DNA was extracted using standard methods, usually from dried blood spots (DBS) submitted on filter cards (CentoCard®) Table . Sanger NPC1, were targeted for analysis [For the CentoMetabolic® panel, the coding regions, 10 bp of flanking intronic sequences, and known P/LP variants (coding and non-coding) of the selected genes, including analysis . Data ananalysis , 18.NPC1 variants were classified according to the published American College of Medical Genetics and Genomics (ACMG)/Association for Molecular Pathology (AMP) guidelines as P, LP, and variant of unknown significance (VUS) [ce (VUS) , 20.The regional origin of the patients was categorized into main geographical regions, namely, Europe, Asia, Africa, Middle East, Latin America, and North America. This corresponded to the origin of the requesting clinic/institution.P/LP variants were grouped into different classes, based on the impact of the mutations at protein function, namely, (1) LoF, (2) missense/conservative, and (3) unknown coding effect. The LoF group included frameshift, canonical splice site, nonsense, and disruptive large deletion/duplication variants. Missense and in-frame deletion/duplication variants were grouped into the missense/conservative category, while synonymous and other intronic variants were grouped into the unknown coding effect group.Patients’ phenotype was evaluated using HPO terms, age at diagnosis, and biomarker levels. The HPO terms were classified into main groups: neurological (11 HPO terms), visceral (8 HPO terms), eye related (3 HPO terms), and others (8 HPO terms) (Supplementary Table 24H50O7N2P—legacy name lyso SM-509) [The quantitation of the biomarker PPCS (C SM-509)  in DBS wClinical characteristics of the patients identified in the CENTOGENE Biodatabank were summarized as proportions for categorical variables and medians for continuous outcomes.Comparisons of patients and variant characteristics per demographic variables were done using proportions, and the significance was tested using chi-square test.p values for pairwise comparisons were estimated after correcting for multiple testing. All shown p values have been adjusted for multiple testing.The phenotypes  were compared with main variables, namely: geographical regions, impact of the variant , and variant frequency for the most common variants. The difference between the phenotypes and the variables was tested using the Kruskal–Wallis test, and https://www.r-project.org/).Finally, multivariable regression analyses were used to test the associations between main HPO phenotype outcomes  and the most common variants using logistic regressions with the main outcome being presence = 1; absence = 0 . Here the associations were represented as the probability (odds ratios) of having a clinical outcome (HPO terms) for patients with a variant vs. patients without the variant. Linear regressions were used to test for associations between age of diagnosis and biomarker levels with the most common variants. Here the significant association is represented as the increase in the mean unit in patients with a variant vs. patients without that variant. The analyses were done using R package v.4.1.1 , compared to female patients . For the rest , gender information was not included in the requisition form. Information regarding age at onset of symptoms was not provided by most referring doctors. Thus, the age at diagnosis was recorded as a proxy of disease subtype and severity. Most patients were younger than 5 years, corresponding to the neonatal/infantile forms , and the median age at diagnosis was 10.6 years (range 0–65 years).The demographics of the cohort of 602 NPC1 patients are described in Table N = 190, 32%) and Latin America , followed by Africa  and Europe   or gene panels  were requested  Fig. . For mos% Fig. . HPO terms were retrieved from the CENTOGENE Biodatabank in 456 patients. HPO terms had been previously extracted and curated using the clinical information provided in the requisition form and registered in the LIMS by a dedicated team.The median biomarker level was 1650 ng/ml blood (IQR: 1077-2816). Almost all patients had pathological biomarker levels (cut-off at 655 ng/ml), except for 33 patients who showed normal levels of the biomarker. Among these cases there was no overrepresentation of a particular variant or variant type, geographical origin, or age/year at diagnosis. We noted that approximately half of these cases had borderline upper levels of biomarker (576–655 ng/ml). Repetitions could not be done at a later age or using a new sample.We identified 830 P/LP variants in 602 patients from our Biodatabank. Of these, 287 were unique and 73 were novel variants (previously unpublished). Most of the novel variants were detected in patients from the Middle East and Latin America Fig. . Figure NPC1 variants per geographical region of origin of the patients. The most common variants per region are present in Fig. We looked at the prevalence of the variants per geographical origin of the patients. There were 73 variants (12%) for which the origin was unknown. The most common disease-causing variant of the complete dataset p.P1007A was also the most common variant detected in patients from Europe. The Top2 variant p.A1035V was the most frequent variant detected in patients from Latin America, and the Top3 p.S954L was the second most commonly detected in patients from Europe. Two of the most common variants in patients from Africa were unique to this continent and accounted for 11% of all variants in this continental region , while 32% of the remaining variants were unique. There was an evident clustering of https://hpo.jax.org/app/browse/gene/4864) . The second most reported clinical term was neurodevelopmental abnormality , followed by abnormality of movement  and abnormality of eye movement . We further classified the terms into neurological, eye, visceral abnormalities, and “other” for further analysis . As expected, patients with LoF variants had a significantly earlier age at diagnosis compared to patients with other types of variants  and to patients with unknown effect variants  and biomarker levels also lead to significant differences. The patients with LoF variants had higher biomarker levels compared to patients with missense variants . Therefore, biomarker PPCS levels were inversely proportional to age at diagnosis  for Africans, 3 years (IQR: 0.5–6) for Asians, 4 years (IQR: 1–10) for patients from the Middle East, 9 years (IQR: 4–24 years) for Latin Americans, and 14 years (IQR: 6–31 years) for Europeans . We focused on three main group of symptoms, namely having eye abnormalities , neurological abnormalities (yes: 491 no: 117), and visceral abnormalities .To better understand the relationship between p = 0.0029, Supplementary Table p value = 0.0017, Supplementary Table The variant p.I1061T (Top5) was strongly associated with eye-related abnormalities , whereas visceral abnormalities were associated with an earlier age at diagnosis  , p.A1035V (Top2), p.S954L (Top3), p.R1186H (Top4), and p.I1061T (Top5) were associated with age at diagnosis and biomarker values. We found that patients with the variant p.P1007A (Top1) had a significant later age of diagnosis of 13 years when compared to patients without the variant. Patients that have the p.S954L variant (Top3) had an age of diagnosis 22 years later than patients without the variant Table . There wNPC1 variants) in our laboratory over the past 15 years. To date, this is so far the largest dataset described for NPC1 disease. Other large cohorts include a study from the United Kingdom that describes 114 patients with NPC1 causative variants [NPC1 variants [We describe a cohort of 602 NPC1 patients from 47 countries as identified in the CENTOGENE Biodatabank. The patients were genetically diagnosed .Within our study, the genetic diagnosis of the patients was done using a combination of genetic and biomarker testing whenever blood samples were provided. We confirmed the high allelic heterogeneity of n = 157) being unique to a single patient, we identified six recurrent variants. The most frequent variant in our cohort was p.P1007A, which was detected in 48 patients (5.8%). This is a known pathogenic variant which leads to the “variant” biochemical phenotype [NPC1 variant [NPC1 disease alleles [Although most of the variants identified in the database were rare, with ~21%  had higher biomarker levels. These patients with a young age at diagnosis and higher biomarker levels also more frequently presented LoF variants and a visceral (abdomen/liver) phenotype. This suggests that the biomarker PPCS could serve as an indicator of disease severity. This observation is in line with a previous report of 36 NPC1 patients with 73 measurements analyzed and showed that the biomarker PPCS significantly correlated with the annual severity increment score . RecentlThe compilation of variants in this cohort together with the demographic, clinical, and biomarker characteristics allowed us to identify interesting patterns. Traditionally, genotype to phenotype analysis is patient-centric . In this study, we dissected the patients’ phenotypes using the HPO terms related to each patient’s clinical presentation. Although the age at diagnosis of most patients in the cohort is below 5 years of age (44%), 23% of the patients were between 5–18 years old at the time of the diagnosis and 15% of the cohort was diagnosed during adulthood—demonstrating that the known clinical and age-related variability of the NPC1 phenotype is represented in our cohort.NPC1 variant p.I1061T  was associated with an eye phenotype  and detected mainly in patients from Latin America. This variant has been reported to account for 20–25% of alleles in patients diagnosed in France [The variant p.S954L was significantly associated with eye-related phenotypes , to a later age at diagnosis, and to lower biomarker levels Fig. . This con France  and the n France —showing n France , 32. Thin France . Patientn France .NPC1 variants per geographic area , therefore the geographical distribution of patients and variants in our study might be not representative. Unfortunately, we do not have sufficient data related to the age of onset of symptoms and the natural history of the disease, nor do we have clinical information to establish a scale of severity of the disease. Furthermore, we must flag that the clinical assessment for NPC1 patients might greatly differ in different countries due to awareness, availability of diagnostic resources, and training of health care professionals impacting the setting in which children and adults with suspected neurometabolic disorders gained access to molecular testing offers.NPC1 allelic heterogeneity. In addition to the diagnostic value of the biomarker PPCS, which is especially valuable in patients with missense variants, the biomarker might be useful to indicate disease severity/progression. Additionally, we confirmed previous genotype–phenotype associations and established novel genotype-HPO term-biomarker relationships for the most frequent NPC1 variants.In conclusion, we describe the largest and most heterogenous cohort of NPC1 patients published to date, including 73 novel P/LP variants, expanding Supp material


Circular RNAs are highly stable single-stranded circular RNAs and enriched in the brain. Previous studies showed that circRNAs, as part of competing endogenous RNAs (ceRNAs) network, play an important role in neurodegenerative and psychiatric diseases. However, the mechanism of circRNA-related ceRNA networks in postoperative cognitive dysfunction (POCD) has not been elucidated yet. POCD usually occurs in elderly patients and is characterized by hippocampal dysfunction. Here, aged C57BL/6 mice were subjected to exploratory laparotomy under sevoflurane anesthesia, and this POCD model was verified by Morris water maze test. Whole-transcriptome sequencing was performed on the hippocampus of control group (Con) and surgery group. One hundred and seventy-seven DEcircRNAs, 221 DEmiRNAs and 2,052 DEmRNAs were identified between two groups. A ceRNA network was established with 92 DEcircRNAs having binding sites with 76 DEmiRNAs and 549 target DEmRNAs. In functional enrichment analysis, a pathological pattern of POCD was highlighted in the ceRNA network: Abnormal metabolic process in neural cells, including oxygen metabolism, could promote apoptosis and then affect the synaptic function, which may undermine the neural plasticity and eventually lead to changes in cognitive function and other behavioral patterns. In conclusion, this specific ceRNA network of circRNAs–miRNAs–mRNAs has provided novel insights into the regulatory mechanisms of POCD and revealed potential therapeutic gene targets. Circular RNAs (circRNAs) are one of the noncoding RNAs (ncRNAs), that are highly abundant and evolutionarily conserved . Having Perioperative neurocognitive disorders (PND) include postoperative delirium (POD) and long-term postoperative cognitive dysfunction (POCD), and it is one of the most common perioperative complications. The risk of POCD increases in the elderly patients (> 60 years), appearing to be 25–40% . POCD isAmong these pathologies, ncRNAs , together with miRNAs, play important roles in regulating related genes at the transcriptional level. CircRNA-089763 was related to POCD in elderly patients who underwent non-cardiac surgery . We founad libitum). Animal experiments were performed following the guide for the care and use of laboratory animals, and the protocol was approved by the local biomedical ethics committee (No. LA2018085).C57BL/6 female mice (18-month-old and weighing 25–30 g) were used in this study. The mice were acclimatized for 2 weeks on a standard housing condition  of sevoflurane for mice was 2.4–2.7% (2 for the same periods without anesthesia or surgery. The hippocampus tissue of the mice was dissected and stored at –80°C at 48 h after the surgery in both control and surgery groups.The aged mice were randomly assigned to control and surgery groups. The surgery group received 2.5% sevoflurane anesthesia with 50% fresh air and 50% O2.4–2.7% . The mic2.4–2.7% . A longi2.4–2.7% . The mic2.4–2.7% . The mic™ to generate paired-end reads (150 bp in length). The quality of RNA-seq reads from all the brain tissues was checked using FastQC . Given the heterogeneity of different batches of data, we just used our own sequencing data rather than combined analysis with online database.Library construction was performed according to the Illumina sample preparation for RNA-seq protocol. The mRNA was enriched by magnetic beads with Oligo (dT) after the samples were qualified. When the enrichment was complete, the mRNA was interrupted into short segments with the addition of a fragmentation buffer. Subsequently, double-stranded cDNA was synthesized by reverse transcription using 6-base random primers. The purified double-stranded cDNA was subjected to terminal reparation, single nucleotide A (Adenine) addition and serial sequencing. The fragment size of double-stranded cDNA was selected by an AMpure XP bead , and the selected double-stranded cDNA was subjected to PCR enrichment to construct a cDNA library. The RNA-seq library for each sample was constructed and sequenced  based on the protocols of Illumina HiSeqTM2500/MiSeqp < 0.05 as the definition of DEG. Read count data were standardized with TMM, and the differences in expression were analyzed by DEGseq (v1.34.0). The overall distribution of the differential genes was shown by scatter plots.Differential expression analysis was performed using the HTSeq v0.5.4p3 to identify differentially expressed circRNAs, miRNAs and mRNAs . To better indicate postoperative changes of circRNA, miRNA and mRNA, we just used Considering miRanda has a high accuracy in predicting miRNA-target interactions for brain study in mice model, we used miRanda database  to prediin silico PCR. Amplification was carried out with an initial denaturation step at 95°C for 2 min, followed by 45 cycles of 95°C for 10 s, 55°C for 30 s and 60°C for 30 s, then 65°C for 2 min in 10 μl reaction volume. All reactions were run in duplicate.Quantitative real-time PCR (qPCR) was performed on the CFX96 Real-Time PCR Detection System . Amplification mixture consisted of PowerUp SYBR Green master mix , 10 μM forward and reverse primers  and approximately 1.5 μl of cDNA template. Primer sequences were obtained from the literature and checked for their specificity through The Morris water maze (MWM) test  was used to assess the spatial learning and memory of mice after surgery. Morris water maze test consisted of a circular tank (120 cm in diameter and 50 cm high) containing water (23 ± 1°C) that is divided into four quadrants and a platform (10 cm in diameter) located 1 cm below the water in the target quadrant. In the place navigation test, the mice were placed in one quadrant facing the wall of the maze and allowed to explore for the hidden platform for 90 s in each trial . The time to locate the submerged plat form was recorded (defined as the escape latency). If the platform was not found within 90 s, the mice were guided to the platform, where they stayed for 15 s. Mice underwent daily testing in the water maze from day 1 to 5 after surgery. On postoperative day 6, the submerged platform was removed from the water maze and a spatial probe test was performed for 90 s. The swimming speed, escape latency, times of platform crossing, and the time spent in target quadrant were recorded by a video camera.t-test was used to identify significant differences between the two groups. The p value less than 0.05 was considered statistically significant. The correction for multiple testing was applied using the Benjamini–Hochberg method and genes with an FDR < 0.05 were used in downstream pathway enrichment analyses. Enrichment of pathways from KEGG and GO Biological Processes was assessed with Fisher’s exact test, followed by multiple testing correction with the Benjamini–Hochberg method.The statistical calculations were performed with Graphpad Prism 7.0 software. Quantitative data were presented as the mean ± standard deviation (SD). Non-paired double-tailed Student’s p < 0.05; p < 0.05; Hippocampus samples from 12 aged mice, including 6 in surgery group and 6 in control group, were enrolled in the study. A total of 3,532 circRNAs were analyzed, and 177 DEcircRNAs (85 up-regulated and 92 down-regulated) were identified between surgery and control groups  or down-regulated  and ENSMUST00000041357 (Lrg1), while down-regulated in ENSMUST00000174874 (Cobl) and ENSMUST00000106233 (Baiap2) in surgery group compared with control group. Among the circRNAs, mmu_circ_0001231 (Focad), mmu_circ_0001427 (Limk1) and mmu_circ_0000114 (Mpzl1) showed down-regulation, and mmu_circ_0001661 (Csmd1) showed up-regulation in surgery group . The top terms with repetitive significance were screened and ranked by the number of related genes in the histogram.The potential biological roles of the 2,052 DEmRNAs were predicted through GO annotation and KEGG pathway enrichment analysis, revealing an enrichment of 1762 GO terms  and 15 KEGG pathways  included ECM-receptor interaction, glutamatergic synapse, PI3K-Akt signaling pathway, dopaminergic synapse, focal adhesion, calcium signaling pathway ; novel_circ_0015901 ; novel_circ_0007300, novel_circ_0006219 (mmu-miR-370-3p). These related DEcircRNAs were all down-regulated like Kmt5c. Dlg4, Map6, Hdac7, Marveld2 and Cobl. Among them, Dlg4 encodes a major synaptic protein. PSD95 clusters glutamate receptors and is critical for plasticity. Dlg4 was down-regulated in postoperative period. In the ceRNA network, Dlg4 was negatively regulated by mmu-miR-665-3p. This DEmiRNA interacted with mmu_circ_0000114, novel_circ_0019750, and novel_circ_0009314. These related DEcircRNAs were all down-regulated like Dlg4.Ryr3, Klf10, Dlg4, Osbpl5 and Hdac7. Among them, Ryr3 controls the intracellular Ca2+ levels and reproduces the core phenotypes of brain aging  ; novel_circ_0018415, novel_circ_0024225 (mmu-miR-674-5p). These related DEcircRNAs were all down-regulated as Hdac7 did. Dlg4, Astn1, Ano1, Olfm2 and Synpo. Among them, Ano1 encodes a member of Ca2+ activated Cl− channels (CaCCs) that are critical for synaptic potential and neuronal signaling, which affects learning and memory processes  . Ryr3 waon death . Hdac7 wrocesses . Ano1 wan = 12). The place navigation test began at 1 day after surgery, and during five training days, the swimming speed maintained constant and showed no significance between two groups, indicating the intact locomotor activity after surgery . Second, this study just provided ceRNA network and the direction for circRNA related pathological processes. For specific pathway or therapeutic targets, intervention studies are still necessary in the future. Third, there are multiple perioperative factors for the occurrence of POCD including anesthesia, surgical trauma, and baseline status of the brain, this study did not analyze their influence, and the conclusion was relatively limited.This study identified DEcircRNAs, DEmiRNAs, and DEmRNAs in the hippocampus of aged mice after surgical intervention and sevoflurane anesthesia, and indicated that circRNAs could take part in the occurrence and development of POCD through ceRNA network and protein-coding gene regulation, which are involved in a pathological pattern including metabolic process, neural plasticity and apoptosis. These results provide novel insights into circRNA-related regulatory mechanisms for POCD, and reveal the potential therapeutic gene targets.https://doi.org/10.5061/dryad.z612jm6gn). The temporary sharing link to download can be found here: https://datadryad.org/stash/share/XxL_uy1jIqeuap9KVWGUjBqHZ3lxdGvPs2EBNy3aPYI.The results of differential expression analysis , gene enThe animal study was reviewed and approved by Biomedical ethics committee of Cancer Hospital, Chinese Academy of Medical Sciences and Peking Union Medical College (No. LA2018085).MZ performed the experiments, analyzed the data, and wrote the manuscript. ZS contributed to experiments, data analysis and manuscript revision. YQ contributed to data analysis. YZ and WX contributed to data analysis. BZ, QW, and LW contributed to data analysis and manuscript revision. SL and YC contributed to the manuscript revision. TX contributed to the project supervision and manuscript revision. CN and HZ designed the project, supervised the experiments, drafted and revised the manuscript. All authors contributed to the article and approved the submitted version.This work was supported by the National Natural Science Foundation of China , Talent Project of National Cancer Center/Cancer Hospital Chinese Academy of Medical Sciences for CN, and Beijing Hope Run Special Fund of Cancer Foundation of China (No. LC2020A01).The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest.All claims expressed in this article are solely those of the authors and do not necessarily represent those of their affiliated organizations, or those of the publisher, the editors and the reviewers. Any product that may be evaluated in this article, or claim that may be made by its manufacturer, is not guaranteed or endorsed by the publisher.


Vibrio spp. in aquatic ecosystems. To determine the impact of environmental factors on occurrence of pathogenic Vibrio spp., samples were collected in the Chesapeake Bay, Maryland, during 2009 to 2012 and 2019 to 2022. Genetic markers for Vibrio vulnificus (vvhA) and Vibrio parahaemolyticus  were enumerated by direct plating and DNA colony hybridization. Results confirmed seasonality and environmental parameters as predictors. Water temperature showed a linear correlation with vvhA and tlh, and two critical thresholds were observed, an initial increase in detectable numbers (>15°C) and a second increase when maximum counts were recorded (>25°C). Temperature and pathogenic V. parahaemolyticus (tdh and trh) were not strongly correlated; however, the evidence showed that these organisms persist in oyster and sediment at colder temperatures. Salinity (10 to 15 ppt), total chlorophyll a (5 to 25 μg/L), dissolved oxygen (5 to 10 mg/L), and pH (8) were associated with increased abundance of vvhA and tlh. Importantly, a long-term increase in Vibrio spp. numbers was observed in water samples between the two collection periods, specifically at Tangier Sound (lower bay), with the evidence suggesting an extended seasonality for these bacteria in the area. Notably, tlh showed a mean positive increase that was ca. 3-fold overall, with the most significant increase observed during the fall. In conclusion, vibriosis continues to be a risk in the Chesapeake Bay region. A predictive intelligence system to assist decision makers, with respect to climate and human health, is warranted.Incidence of vibriosis is rising globally, with evidence that changing climatic conditions are influencing environmental factors that enhance growth of pathogenic IMPORTANCE The genus Vibrio includes pathogenic species that are naturally occurring in marine and estuarine environments globally. Routine monitoring for Vibrio species and environmental parameters influencing their incidence is critical to provide a warning system for the public when the risk of infection is high. In this study, occurrence of Vibrio parahaemolyticus and Vibrio vulnificus, both potential human pathogens, in Chesapeake Bay water, oysters, and sediment samples collected over a 13-year period was analyzed. The results provide a confirmation of environmental predictors for these bacteria, notably temperature, salinity, and total chlorophyll a, and their seasonality of occurrence. New findings refine environmental parameter thresholds of culturable Vibrio species and document a long-term increase in Vibrio populations in the Chesapeake Bay. This study provides a valuable foundation for development of predicative risk intelligence models for Vibrio incidence during climate change. Vibrio  infrastructure  and thermostable direct-related hemolysin (encoded by trh), which potentially carry out lysis of human erythrocytes  which encodes phospholipase A2 (V. parahaemolyticus and is commonly employed as a marker for species detection and identification (V. vulnificus causes infection in humans are more complex (vcg) (V. vulnificus hemolysin A gene (vvhA) has been established as reliable species-specific biomarker , in nutrient cycling, and in other biogeochemical processes  , the extex (vcg) . Howeveriomarker . Since Vrocesses –51, theyV. cholerae and noncholera vibrios have various routes of environmental transmission, primarily attributed to the ability of V. cholerae to thrive in freshwater as well as coastal ecosystems  (P < 0.05) (a (Chl a), pheophytin, and active Chl a, the highest values were recorded at the Upper Patuxent River (UPR) station, while the lowest values were recorded at TS . For other environmental parameters, little variation was observed among the other stations, but values varied by season at each station.Between June 2009 and August 2012, a total of 111 total field observations were recorded from the Chester River (CR) and Tangier Sound (TS), and between April 2019 and August 2022, 86 total field observations were recorded from 12 stations . A summa < 0.05) , with th < 0.05) . For totV. parahaemolyticus and V. vulnificus in various sample types  . Both vvhA and tlh were detected at a higher abundance than trh and tdh . Between 2019 and 2022, concentrations of tlh and vvhA showed no overall difference with respect to sampling station  .Direct plating and DNA colony hybridization (DPCH) was used to determine incidence and abundance of le types . Overall < 0.05) . Overall > 0.05) , and tlhV. parahaemolyticus and V. vulnificus may have changed over time, DPCH results for water samples were compared between the two collection periods. Across all stations, no overall difference was observed between the two collection periods for vvhA, but tlh concentrations increased significantly during the 2019–2022 collection period compared to 2009 to 2012, whereby median log1p, i.e., [log(x + 1)], values increased from 0.4 to 2.4  and mean positive values increased from ca. 5 CFU/mL to ca. 18 CFU/mL  and mean positive values increased from ca. 18 CFU/mL to ca. 37 CFU/mL , with the greatest increase observed during the fall, when median log1p values increased from 0.55 to 2.67 and mean positive values increased from ca. 3 CFU/mL to ca. 22 CFU/mL.To evaluate how concentrations of 8 CFU/mL . With re7 CFU/mL . SimilarvvhA and tlh from the same station between collection periods. Water samples from the TS station showed no overall difference for environmental parameters, including temperature, salinity, conductivity, TDS, DO, and total Chl a, between the two periods . Like the comparison of all stations, no statistical difference was observed for the concentration of vvhA, but an increase was observed for tlh, in which median log1p values increased from 0.41 to 2.43  and mean positive values increased from ca. 5 CFU/mL to ca. 18 CFU/mL , with the greatest increase observed during the fall, when median log1p values increased from 0.55 to 2.67 and mean positive values increased from ca. 2 CFU/mL to ca. 22 CFU/mL  . Compared to tlh, negative results were more frequent when DPCH was used to quantitate trh and tdh populations in water, oyster, and sediment samples from both stations between 2009 and 2012. Both tdh and trh were detected during warmer months, with higher detection in oyster and sediment samples. As for tlh and vvhA, the trh and tdh detection rates were highest for sediment, followed by oyster and water. Pathogenic V. parahaemolyticus (trh and tdh) were detected more frequently during the winter months in sediment than in water and oysters. With exception of trh in oysters (tau = 0.4), the correlation with temperature was not strong. A similar pattern of seasonality was observed for samples collected between 2019 and 2022  than water samples   . Howeverand 2022 , where t = 0.56) .a were determined to be strong predictors of increased abundance of both vvhA , with the greatest association observed for vvhA occurrence in oyster samples . Incidence of both trh and tdh was associated with temperatures of >20°C in oyster and water samples, but moderate abundance of these markers was detected occasionally at lower temperatures in sediment samples compared to water and oyster samples.With respect to seasonal temperature, occurrence of both  samples . As tempbe >15°C . A secontlh and vvhA was nonlinear, demonstrative of a downward curve . Moderate abundance of vvhA was associated with salinity ranges between 3 ppt and 12.5 ppt at CR and ranges between 10 ppt and 17 ppt at TS. Differences in salinity between stations were more prominent for tlh in water  than in oyster and sediment . For samples collected between 2019 and 2022, salinity ranges associated with moderate abundance were more similar among sampling stations, with exception of tlh detection in water at Choptank River (CHR) and UPR stations (6 to 12 ppt) compared to TS (12 to 17 ppt), whereby salinity ranges associated with moderate abundance were observed between 7 and 17 ppt for both tlh and vvhA .The overall relationship between salinity and abundance of both rd curve . Evaluat for tlh . Howevera and detection of tlh and vvhA was observed as a nonlinear downward curve  and active Chl a (10 to 20 μg/L) were observed to be associated with tlh and vvhA detection at increased abundance.As for salinity, the relationship of total Chl rd curve . Across  28 μg/L . HoweverVibrio abundance was consistent across collection periods and location, observed as a nonlinear relationship with an acceptable range for occurrence of genetic markers. For all markers, pH ranges between 7 and 9 were associated with moderate detection, and pH values closer to 8 were generally associated increased abundance . DO ranges between 4 mg/L and 15 mg/L were associated with moderate detection of vvhA and tlh. The DO range for increased abundance of tlh and vvhA was observed between 5 mg/L and 8 mg/L, which was also within the range for presence of pathogenic V. parahaemolyticus markers. Ranges for conductivity (15 to 22 mg/L) were associated with increased abundance of vvhA and tlh during both periods. Between 2009 and 2012, TDS ranges (10 to 15 g/L) were associated with increased abundance, while slightly higher values were observed (15 to 25 g/L) during the 2019–2022 collection period.The relationship of other environmental parameters and Vibrio spp. thrive in warm water and moderate salinity , natural calamity , and/or social disruption and civil unrest . For example, the risk of cholera can now be predicted at the scale of available satellite observation and favorable environmental conditions , 63. Theirements  and assoirements . Other may Water  and the p Viewer  developeV. cholerae and noncholera Vibrio spp. in the ocean and estuarine environments through cross-sectional studies, including long-term retrospective work  state  state –78, in w) state –. V. paranditions , 81. Intnditions , 82–84. richment . The resV. parahaemolyticus and V. vulnificus in various sample matrices were quantified using DPCH  in the environment  were observed in the middle of July. Subsequently, V. parahaemolyticus numbers stayed around 103 CFU/100 mL until the middle of October when the water temperature dropped below 15°C  for 2019 to 2022 compared to 2009 to 2012, with the greatest increase observed during the fall for both tlh (ca. 7-fold) and vvhA (ca. 2-fold). Similarly, water samples collected at TS, the station consistently sampled during both the 2009–2012 and 2019–2022 collection periods, showed increases in tlh during summer, fall, and winter, with the greatest increase in the fall. However, no significant difference was observed for vvhA at TS between collection periods, which might be due to the fact that V. vulnificus has been found to be concentrated in the middle bay, upper bay, and western estuaries compared to stations in the lower bay  and ca. 85% (2019 to 2022) of the oyster samples were positive for tlh. Values for oyster samples evaluated by weight during 2009 to 2012 were low, according to United States Food and Drug Administration (FDA) guidance for V. parahaemolyticus (<104 CFU/g) in ready-to-eat foods  compared to volume (2019 to 2022). Thus, a direct quantitative comparison could not be made. However, the rates of detection using genetic markers were similar; e.g., the study reported in reference at foods . Overall reports .Vibrio abundance, in agreement with findings of previous studies  and chlorophyll was found to be nonlinear (a (5 to 25 μg/L), namely, pheophytin (4 to 8 μg/L) and active Chl a (10 to 20 μg/L). With respect to pathogenic V. parahaemolyticus (trh and tdh), a peak density was observed around 10 μg/L. However, chlorophyll as a proxy for zooplankton abundance would benefit by including specific lag times, e.g., 1 month, to account for a subsequent increase in zooplankton , and sediment samples were collected at two stations, i.e., CR and TS, totaling 111 sampling events. The Chesapeake Bay, an inlet of water enclosed on three sides by land, is the largest estuary in the United States. CR is a major tributary in the northern portion of the Chesapeake Bay comprising brackish water, while TS is an inlet of the larger Atlantic Ocean with a more saline water content and is located further south. Between April 2019 and August 2022, water and oyster samples were collected from 12 stations throughout the Chesapeake Bay, totaling 85 sampling events  was done twice per month during warmer months (June through August) and once per month the rest of the year. Between 2019 and 2022, sampling (water and oyster) was done weekly during the warmer months and twice per month during the rest of the year. However, between March 2020 and July 2020, no samples were collected due to laboratory restrictions associated with coronavirus disease 2019 (COVID-19). Water (12 L) was collected 0.3 m below the surface using a Van Dorn water sampler  and stored in a sterile Nalgene carboy . The carboy was rinsed three times with sample water from the site prior to collection. Up to 30 oysters were collected by dredging and stored in clean double-zipper freezer bags. Sediment (ca. 100 g) was collected using a stainless-steel scoop and stored in sterile Nalgene bottles . Samples were transported to the laboratory in a cooler with ice packs, ensuring that samples did not come in direct contact with the ice packs. Temperature of the coolers was monitored to ensure that it did not reach above 8°C during transport (<4 h) using a LogTag single-trip temperature alert indicator . The temperature of the water samples was recorded upon arrival at the laboratory, and samples were stored at 4°C until processing (<12 h). It is worth noting that internal analysis of split samples processed immediately  and following refrigeration for 12 h showed no significant change in the number of culturable bacteria detected in homogenized oyster tissue or water samples at a volume of >10 L, using methods detailed below.a was measured by high-performance liquid chromatography. Briefly, volumes up to 250 mL of water were filtered using 25-mm glass microfiber filters  and stored at −80°C. For samples collected between 2009 and 2012, concentrations of total Chl a were measured in methanol extracts on a Cary model 50 UV–visible-light spectrophotometer, as described previously  and pheophytin were measured in acetone extracts on a Shimadzu UV 2401PC spectrophotometer following University of Maryland Center for Environmental Science (UMCES) standard operating procedures for fluorometric determination of Chl a in waters and sediments of fresh/estuarine/coastal areas . Air temperature was recorded at the time of collection. Total Chl eviously , 112. Foal areas , modifieal areas  and Ameral areas . Chl a aBacteriological Analytical Manual for food sampling/preparation of sample homogenate  to enumerate V. parahaemolyticus and V. vulnificus, respectively. Following incubation at 37°C for 16 to 18 h, Whatman 541 ashless filters  were used to lift colonies from each plate. Filters were probed using alkaline phosphatase-conjugated oligonucleotide probes  specific for V. parahaemolyticus (tlh) or V. vulnificus (vvhA); samples collected between 2009 and 2012 were also screened for V. parahaemolyticus virulence markers (trh and tdh). Probes, listed in Vibrio bacteria, 1 mL was spread onto T1N3 or VVA during each hybridization. Cultures of V. parahaemolyticus , and V. vulnificus (ATCC 27562 [vvhA+]) were prepared under standard growth conditions in Luria-Bertani broth at 37°C overnight with aeration, and 1 μL was spotted onto T1N3 agar or VVA three times for each hybridization to serve as positive and negative controls, respectively.Methods to enumerate eviously , 90, 117x + 1)]. To evaluate the relationships of environmental parameters and DPCH results, surface recordings were used for water samples and bottom recordings were used for oyster and sediment samples. Both individual stations and pooled data were evaluated for both collection periods. However, since samples collected between 2009 and 2012 were representative of >50 sampling events from each of two stations, 15 sampling events were randomly selected from both CR and TS using the “sample” command from the base R software package. To ensure consistency of subsampling across data analysis, the seed was set to seven, which was chosen by random number generation. To test for significance of the observed differences of means between two groups, Wilcoxon test was used, while one-way ANOVA was used to compare differences of means among more than two groups. Linear regression and Kendall’s tau correlation methods were used to quantify the relationship of DPCH results with temperature. A nonparametric regression model, i.e., locally weighted smoothing , was used to visualize the relationship of DPCH results with environmental parameters lacking linear correlation. Box plots were used to summarize distributions by indication of interquartile range. Scatterplots were used to visualize DPCH results relative to environmental parameter values. Normalized kernel density estimates, i.e., a smoothed version of the histogram, were used to visualize the count of samples demonstrating >10 CFU relative to each environmental parameter value.Statistical analysis was done using R v.4.2 , and fig


GIGER MD device applies a biofeedback method through stimulated coordinated rhythmic and dynamic movements of the trunk and extremities in an anti-gravity position, thus helping to regain lost motor functions.In this article, the performance of the GIGER MD device was evaluated in 36 children with neurogenic bladder measuring gait speed, voiding bladder capacity, deviation from the age-adjusted bladder capacity , and urinary incontinence.per day), and the 20-meter motor gait speed increased by 14.26% (from 23 to 19 s).Children using the GIGER MD device had an increase in voiding bladder capacity  with a subsequent median decrease in median age-adjusted bladder capacity by 45.50% (median deviation before was 36% vs. 16% after treatment). The number of urinary incontinence episodes also reduced by 55.57% (7–3 episodes Children who follow the GIGER MD therapy regularly for a period of 6 months show that CNS functional damage can be significantly improved. Rehabilitation of children requires a multidisciplinary approach that uses brain neuroplasticity to stimulate and develop normal motor patterns of posture and movement . GIGER Mvia catheter bags. Children with nocturia were woken by their parents 1 and 4 h after falling asleep and offered to urinate. Age-adjusted bladder capacity was estimated using Koff’s equation: expected capacity (mL) = [age + 1] ×30 , while catheterized children’s volumes were measured p-values ≤0.05 were acknowledged as statistically significant. Statistical analysis was performed with the program GraphPad Prism 8.4.3.686.The following variables were measured before and after GIGER MD therapy . Data are expressed as mean, standard deviation, median, and interquartile range (IQR). The difference between paired variables was analyzed using a two-sample paired (Wilcoxon) signed rank test. The Spearman correlation coefficient was applied to assess the strength of a monotonic relationship between paired variables . A correA total of 36 children were enrolled in the study spanning three to 6 months. 25/36 children (A) had a gait assessment, while 36/36 (B) underwent voiding bladder capacity measurement estimation and urinary incontinence frequency. 11/36 children had no gait assessment. There was no difference in training between A and B groups.p < 0.0001, Z-statistic 3.945). The results are shown in The first group of children (A) had an age mean of 12.51 (+/− 4.1) years with a median of 12.33 years (9.792–14.50) and a male-to-female ratio of 1:2.1. The youngest child in the group was 7 years of age. The average group gait speed median before treatment measured 23 s (IQR 19.5–25), while after treatment, the median speed reduced to 19 s (IQR 15–22.5). The 20-meter gait speed after treatment increased by 14.26%, Wilcoxon signed rank test . The results are shown in The second group of children (B) had an age mean of 12.15 +/− 4.51 years with a median of 11.34 years (IQR 8.790–14.19) and a male-to-female ratio of 1:2.17. The average group voiding bladder capacity median before treatment measured 200 (IQR 165–250) milliliters, while after treatment, the median volume increased to 250 (IQR 200–300) milliliters. The voiding bladder capacity after treatment increased by 33.79%, Wilcoxon signed rank test . The results are shown in The average median deviation from the Koff scale before treatment measured 36 (IQR 24–54.5) percent, while after treatment, the median deviation decreased to 16 (IQR 9–39) percent. After treatment, the deviation from the Koff formula decreased by 45.40%, Wilcoxon signed rank test  per day. Episodes of urinary incontinence were reduced by 55.57%, Wilcoxon signed-rank test . The results are shown in The average median urinary incontinence episodes measured 7 (IQR 4–9) p 0.043) and had a higher deviation from the Koff scale  vs. children with no gait assessment. There were no statistical differences between other parameters (Mann–Whitney U-test).Children with gait assessment had reduced voiding bladder capacity  shows a strong negative correlation to the number of rotations per week. The voiding bladder capacity (ρ = 0.46) shows a moderate positive, while simultaneously, the decrease in deviation from the Koff scale shows a moderate negative (ρ = −0.51) correlation habilitation procedures, this sub-area has not been definitively resolved so far and belongs to the domain of future answers.Contemporary treatment is now reduced to several recommendations (1) clean intermittent catheterization (2) reflex voiding and bladder expression with Valsalva or Credé (3) condom catheter drainage or indwelling catheters such as urethral catheters or suprapubic tube (4) urinary diversion by ileal conduit if self-catheterization is impossible (5) suppressing neurogenic detrusor overactivity or compliance alteration  (6) aponeurotic suburethral tape or artificial urinary sphincter for sphincter insufficiency (7) Transcutaneous Electrical Stimulation for Neurogenic Bladder Dysfunction (8) resorting to surgery is sometimes necessary either after the failure of non-invasive treatments  . We hereThe best achievement of applying the GIGER MD technique was reducing the number of urinary incontinence episodes by 55.57%. The incontinence rate (leakage episodes/per day) shows considerable improvement in most children ; Table 1The second-best achievement was the improvement of bladder capacity. The moderate positive correlation between bladder capacity and gait speed means that the more rotations in the Giger MD a child makes, the more the bladder capacity (in milliliters) will increase significantly. The moderate negative correlation between the Koff scale and gait speed means that the more rotations in the Giger MD a child makes, the more the Koff scale of bladder size will correspond to the values for age (measured as a percentage) . The modThe third-best is the improvement of gait speed. The strong negative correlation between rotations per week and gait speed means that the more rotations in the Giger MD a child makes, the faster the 20-meter track will be walked by a child , 2. TherApplying the GIGER MD method, achieving a satisfactory improvement in urinary incontinence episodes, gait speed, and bladder capacity in patients with neurogenic bladder is possible. The method is harmless, psychologically acceptable for the patient, and can be applied with other non-surgical and surgical procedures. This is the first study using a biofeedback (GIGER MD) device in patients with neurogenic bladder. Further studies are needed to evaluate the long-term effect of the biofeedback (GIGER MD) device on the neurogenic bladder. According to our experience so far, it takes 3–6 months of regular exercise and further maintenance exercise 2 times a month or more often, depending on the possibilities.The raw data supporting the conclusions of this article will be made available by the authors, without undue reservation.The studies involving human participants were reviewed and approved by Ethical Committee of the University Hospital Centre “Sisters of Charity.” Written informed consent to participate in this study was provided by the participants’ legal guardian/next of kin.AC-R, DMil, and VM: conceptualization. AC-R and VM: methodology. DT: software. DMil, GR, and VM: validation. DT and DMil: formal analysis, and writing—original draft preparation and review and editing. AC-R, DMik, GR, and VM: investigation. AC-R, DMik, and VM: resources. DMik and VM: data curation. DT: visualization. AC-R, GR, and VM: supervision. All authors have read and agreed to the published version of the manuscript.The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest.All claims expressed in this article are solely those of the authors and do not necessarily represent those of their affiliated organizations, or those of the publisher, the editors and the reviewers. Any product that may be evaluated in this article, or claim that may be made by its manufacturer, is not guaranteed or endorsed by the publisher.


The trial was conducted at the Department of Neurology, Haukeland University Hospital, Bergen, Norway. The primary outcome was safety, defined as the frequency of moderate and severe adverse events. Secondary outcomes were tolerability defined as frequency of mild adverse events, change in the whole blood and urine NAD metabolome, and change in the clinical severity of PD, measured by MDS-UPDRS. All 20 participants completed the trial. The trial met all prespecified outcomes. NR therapy was well tolerated with no moderate or severe adverse events, and no significant difference in mild adverse events. NR therapy was associated with clinical improvement of total MDS-UPDRS scores. However, this change was also associated with a shorter interval since the last levodopa dose. NR greatly augmented the blood NAD metabolome with up to 5-fold increase in blood NAD+ levels. While NR-recipients exhibited a slight initial rise in serum homocysteine levels, the integrity of the methyl donor pool remained intact. Our results support extending the dose range of NR in phase II clinical trials to 3000 mg per day, with appropriate safety monitoring. Clinicaltrials.gov identifier: NCT05344404.Nicotinamide adenine dinucleotide (NAD) replenishment therapy using nicotinamide riboside (NR) shows promise for Parkinson’s disease (PD) and other neurodegenerative disorders. However, the optimal dose of NR remains unknown, and doses exceeding 2000 mg daily have not been tested in humans. To evaluate the safety of high-dose NR therapy, we conducted a single-center, randomized, placebo-controlled, double-blind, phase I trial on 20 individuals with PD, randomized 1:1 on NR 1500 mg twice daily  at a dose of 3000 mg daily for 30 days is safe and associated with a pronounced systemic augmentation of the NAD metabolome, but no methyl donor depletion. Current treatments for PD provide partial and temporary symptomatic relief for motor symptoms, but make no impact on the rate of neurodegeneration and disease progression4.Parkinson’s disease (PD) affects 1–2% of the population over 65 years, and is one of the fastest growing neurological disorders worldwide5. NAD, which constantly shuttles between its oxidized (NAD+) and reduced (NADH) state, is a coenzyme required for metabolic reduction-oxidation (redox) reactions, including mitochondrial respiration, which are integral to cellular energy metabolism. In addition, NAD+ is substrate for a number of vital non-redox reactions that continuously degrade the molecule, requiring constant replenishment7.Increasing cellular levels of nicotinamide adenine dinucleotide (NAD) has been proposed as a potential neuroprotective therapeutic strategy for neurodegeneration7. Conversely, increasing the NAD replenishment rate via supplementation of precursors has shown beneficial effects on life- and healthspan in multiple animal models, and evidence of neuroprotection in models of parkinsonism and other neurodegenerative diseases10. NAD-replenishment may target multiple processes associated with PD and other neurodegenerative disorders, including mitochondrial function and energy metabolism12, DNA-repair15, epigenetic regulation16, neuroinflammation18, mitophagy15, and lysosomal and proteasomal pathways18. In this manner, NAD-replenishment may increase neuronal resilience, shielding neurons against multiple forms of disease-associated stress15.NAD levels decline with age, possibly due to multiple and only partially understood processes, and this has been proposed to contribute to age-related diseases, including parkinsonisms19. This was, in turn, associated with altered cerebral metabolism and a mild trend for clinical improvement18. Furthermore, NR was associated with transcriptional upregulation of processes related to mitochondrial, lysosomal, and proteasomal function in blood cells and/or skeletal muscle18.In the NADPARK study, a recent randomized double-blind clinical trial of NAD replenishment therapy with nicotinamide riboside (NR) in PD, we showed that oral intake of 1000 mg NR daily for 30 days significantly augments cerebral NAD levels18. It is possible that the neurometabolic effects of NR are dose-dependent and that higher doses, which remain unexplored to date, may prove more beneficial in terms of both effect size and proportion of responders.These findings suggested that NR may hold promise as a neuroprotective therapy for PD, which warrants further clinical investigation. An important knowledge gap in that regard is that the safety range and optimal dose of NR therapy for PD remain undetermined. In the setting of a potential neuroprotective intervention, it is critical that the given dose is sufficient to achieve the desired effects. While the NADPARK trial showed a robust neurometabolic response to NR, the effect size was variable, and not all NR-recipients responded20. Side effects included increased levels of neutrophils and triglycerides in blood, as well as weight loss and decreased organ weight of liver and kidneys22. Moreover, a dose of 3000 mg/kg/day (corresponding to ca. 29 g/day for a 60 kg adult human) resulted in abnormal function in the liver, kidneys, testes, epididymides and ovaries, with signs of chronic progressive nephropathy and centrilobular hepatocellular hypertrophy21.In preclinical studies on rats, adverse effects appeared with doses beyond 1000 mg/kg/day, which corresponds to approximately 9700 mg/day for a 60 kg adult human33, while three studies used 2000 mg daily36. None of these reported any evidence of toxicity or severe adverse effects. Long term treatment for 5 months with a daily dose of 1000 mg NR also did not reveal any adverse effects33.Most clinical studies with NR in adult humans to date have used up to 1000 mg daily37, and changes of the NAD metabolome in blood and urine. Exploratory outcomes included the between-group difference in the change of serum homocysteine levels and of fasting blood glucose and serum insulin levels.To determine the safety of high-dose NR therapy, we performed a double-blind, randomized phase I trial of 3000 mg NR daily in individuals with PD. The primary outcome of the study was the incidence of treatment-associated moderate and severe adverse events (AEs). Secondary outcomes were the between-group difference in treatment-associated mild AEs, change in the clinical severity of PD, measured by the Movement Disorder Society Unified Parkinson’s Disease Rating Scale (MDS-UPDRS)We show that oral NR treatment at a dose of 3000 mg daily for a 4-week period is well tolerated without moderate or severe adverse events, induces a pronounced augmentation of the NAD metabolome, and may be associated with clinical symptomatic improvement in PD. These findings support the possibility of extending the dose range of NR up to 3000 mg in clinical trials, provided adequate safety monitoring. However, the long-term safety of this dose has not yet been established.p = 0.52). There were no significant differences in the frequency of co-morbid medical conditions . Two participants did not use levodopa and were therefore excluded in the analysis of the time since levodopa. Participants did not fast on visits other than baseline visit 1, V1) and last visit , because this interfered too much with their daily function. One participant in the NR group was removed from the analysis of urea, sodium, potassium and creatinine due to data entry error. Pregnancy screening, with serum human chorionic gonadotrophin (HCG), was negative at baseline for all female participants. The groups were similar at baseline with regards to demographics, clinical safety tests, MDS-UPDRS scores and metabolomic parameters , headache (n = 3/3), tremor (n = 2/2), muscle cramp (n = 2/1), fatigue (n = 2/2), nausea (n = 2/2) and dyspepsia (n = 2/2). All cases of dyspepsia and nausea were resolved at the end of the follow up period (Supplementary Data 38.Forty-two adverse events (AEs) were observed overall, 25 of which were in the NR group and 17 in the placebo group. 9/10 participants in the NR group and 8/10 participants in the placebo group experienced at least one AE  in either group, nor were there significant between-group differences in the mean change for each variable Table . Electrop = 0.007; Placebo: V1: 41.8 ± 16.46 vs V7: 41.8 ± 22.23; mean change 0 ± 9.59; paired t-test, p = 1; Table p = 0.024). To determine which symptoms had improved in the NR group, we performed a post hoc explorative analysis of the subparts of MDS-UPDRS (I-IV). The change in the NR group was primarily driven by MDS-UPDRS part III , which measures the severity of motor symptoms, and to a minor extent by part I , which measures non-motor experiences of daily living. The statistical significance for the changes in the subparts of MDS-UPDRS did not survive multiple testing correction  score between V1 and V7  and placebo (−2.85 ± 98.77 min) groups. This analysis revealed a still significant decrease in total MDS-UPDRS in the NR group  but not in the placebo group  and longer in the placebo group (+28.33 ± 105.58 min). Levodopa strongly influences motor symptoms in PD, and the time of intake commonly varies in response to symptom severity+, NADH, NADP+, NADPH and the reduced (GSH) and oxidized (GSSG) forms of glutathione, using the NADMed assay (see methods). The NR group showed a marked increase in the levels of NAD+ and NADH, which resulted in an overall elevated NAD+/NADH ratio , but the mean of the individual changes between the placebo and NR groups was significantly different , but again, the difference of the means of the individual changes between placebo and NR groups reached significance  metabolomics analyses, with a focus on NAD-related metabolites, as well as untargeted metabolomics analyses (see methods). The NR group exhibited a significant increase in whole blood NADsay Fig. . Multiplsay Fig. . Nicotin40. To investigate this possibility, we assessed related metabolites, including homocysteine (HCy), S-adenosyl-methionine (SAM) and S-adenosyl-homocysteine (SAH), as well as methionine and betaine. Clinical routine blood assays showed a mild, but significant increase in serum homocysteine levels in the NR group , but not in the placebo group , and subsequently remained stable until the end of the study  were unaffected, while betaine, a substrate for betaine homocysteine methyltransferase catalyzing one way of methionine synthesis, decreased slightly in the NR group , and duration of exposure. Therefore, while providing an indication of a dose effect, the results of these studies are not fully comparable.At a dose of 3000 mg daily, NR treatment greatly augmented the NAD-metabolome, including a ~3.7-fold (range 1.8–5.8-fold) increase in whole blood NAD+ levels, the NR-group exhibited a clear increase in the NAD+/NADH ratio, which has been reported to be decreased in PD43. Furthermore, NR supplementation was associated with an increase in whole blood NADP. An elevated NADP pool may be beneficial in several ways. NADP is a major redox factor in anabolic synthesis reactions, but also mediates cellular defense against oxidative stress, is involved in cytochrome P450 dependent detoxification mechanisms, and plays a role in immune response by generating oxidative bursts, among others45. With regard to PD, the reduced form of NADP, NADPH, has been shown to ameliorate MPTP-induced dopaminergic neurodegeneration in cultured cells46, and to exert anti-neuroinflammatory and anti-neurotoxic effects in an MPTP-induced mouse model of PD47. Thus, it is possible that NADP augmentation may contribute to NR-associated neuroprotective effects in PD.In addition to prominently elevated NAD+ by the action of CD38 and other enzymes also abundantly expressed in the brain49, further supports a functional impact of NR-induced NAD augmentation on human metabolism.Finally, the observed increase in ADPR, a signaling molecule derived from NAD33, but this was not seen following treatment with 1000 mg NR for one month. This suggests that the NR-associated HCy increase may be dose- and/or time-dependent. The observed increase in serum HCy levels in our data occurred quickly, during the first three days following commencement of treatment, and levels remained stable thereafter, suggesting that a new equilibrium had been reached. Interestingly, the increase in HCy was only observed in routine clinical serum biochemistry, and not reproduced in whole blood metabolomics by LC-MS. The reason for this discrepancy may be related to the sample material , or to method sensitivity. Notably, the change observed in the serum, although statistically significant, was very mild. The observed increase in serum HCy is not unexpected. The NR-induced augmentation of NAD-metabolism leads to increased production of Nam. This is in turn converted to methyl-nicotinamide (Me-Nam) by the action of nicotinamide N-methyltransferase (NNMT), which transfers a methyl group from the universal methyl-donor S-adenosyl methionine (SAM) to Nam, also producing S-adenosylhomocysteine (SAH). SAH is then further converted to homocysteine as part of the homocysteine-methionine cycle  Hoehn and Yahr score <4 at time of enrollment56. Exclusion criteria were: (i) Dementia or other neurodegenerative disorders at baseline visit, (ii) any psychiatric disorder that would interfere with compliance in the study, (iii) any severe somatic illness that would make the individual unable to comply and participate in the study, (iv) use of vitamin B3 supplementation within 30 days prior to enrollment, and (v) metabolic, neoplastic or other physically or mentally debilitating disorders at baseline visit. Patient recruitment, inclusion, and follow-up was carried out by a GCP certified investigator. The research protocol was approved by the Regional Committee for Medical and Health Research Ethics, Western Norway (379218). Patients were identified and recruited at the Department of Neurology, Haukeland University Hospital, Norway. Written and informed consent was obtained from all participants from investigators in NR-SAFE. No financial or other compensation was offered to participants. This study was conducted according to Good Clinical Practice guidelines. The trial was registered on 25.04.2022 on www.Clinicaltrials.gov, identifier: NCT05344404. The study complies with ICMJE guidelines on reporting of clinical trials.This study design was a phase I, single-center, randomized, double-blinded, placebo-controlled trial, aiming to provide initial evidence of safety and tolerability for NR at a dose of 3000 mg daily. The trial was conducted at the Department of Neurology, Haukeland University Hospital, Norway, from the 29.04.22 to the 01.07.22. The first participant was enrolled on 29.04.22 and the last on 27.05.2022. Inclusion criteria were: (i) age equal to or greater than 35 years and lower than 100 years at time of enrollment, (ii) clinical diagnosis of idiopathic PD according to the MDS criteriawww.viedoc.com, version 4.77.8648.13864). Sex was determined by assigned sex in the electronic patient records. NR was administered twice-daily to increase compliance, given the number of capsules participants were required to ingest, and to ensure the stability of the resulting increase in blood NAD levels. The drug containers were sequentially marked by an independent third party at the research and development department at Haukeland University Hospital. The study drug nicotinamide riboside chloride  and placebo were provided by ChromaDex, USA. Each NR-capsule contained 250 mg of NR. Placebo capsules contained microcrystalline cellulose. The NR and placebo capsules were identical in flavor, color, smell and shape. Drug compliance was determined by self-reporting from participants at study visits and a pill count of remaining medication when providing new study medication and at the end of the study. The Center for Clinical Research, Haukeland University Hospital provided allocation sequence, packaging and labeling of drugs in participant-specific kits. Participants, examining physicians as well as medical- and research staff were blinded for the duration of the trial. The sample size was estimated at 10 participants in each group, based on the homogenous response to NR seen in our previous study18.20 participants  were randomly allocated to receive either 1500 mg NR twice daily or placebo for 4 weeks , using the electronic Case Report Form , moderate (2), severe (3), life-threatening (4) or death (5). The AE relation to the study drug was scored as unrelated (1), unlikely (2), possible (3), probable (4) or definitely (5).At screening for trial entry, candidates underwent physical and neurological examination. Enrolled participants were assessed by physical visits by a movement disorder specialist at baseline , day 7 (V4), day 14 (V5), day 21 (V6) and day 28 (V7). At baseline and day 28 participants underwent additional assessment with the Movement Disorder Society Unified Parkinson’s disease Rating Scale section I-IV (MDS-UPDRS). At all physical visits an electrocardiogram (ECG), vital parameters  and blood samples were obtained. Additional blood samples were taken on day 3 (V2) and day 5 (V3) to assess safety. Flash frozen whole blood samples for metabolite analyses (NADMed and LC-MS) were obtained by collecting blood in EDTA tubes, aliquoting into 250µl aliquots and snap freezing the tubes in liquid nitrogen precisely 2 min after blood was drawn. The participants were interviewed by telephone consultation by a study nurse on day 3 and day 35 to screen for additional adverse events. Participants were instructed to take their anti-Parkinson drugs as they normally would and no changes were made to anti-Parkinson drug treatment during the trial. Adverse events (AEs) were recorded by the investigating physician. These were then classified by severity according to the Common Terminology Criteria for Adverse Events v5.0 (CTCAE)Analysis of clinical laboratory values from blood samples was performed by the Department of Medical Biochemistry and Pharmacology (MBF) at Haukeland University Hospital, Bergen, Norway.www.nadmed.com). NAD and glutathione metabolites were extracted from frozen blood in a single step using a proprietary extraction procedure, and each metabolite was measured individually using optimized cyclic enzymatic assays with colorimetric detection. For stability, the NADMed extraction method utilizes an extraction solution representing a non-buffer complex mixture of organic solvents in water. Samples are injected into a pre-warmed extraction solution to force protein unfolding and release non-covalently bound metabolites into solution. All pH and redox sensitive metabolites remain stable, including reduced and oxidized glutathione. After protein removal, supernatants are used for separate measurements of total GSH and GSSG. GSH is determined as the difference of total GSH pool and GSSG values.Analysis was carried out by NADMed .g and 4 °C, 900 µl of extract were added into a Phree filter, centrifuged for 10 min at 1800 × g and 4 °C. 250 µl of the filtrate were transferred into two high-recovery LC-vials  and samples dried under a flow of nitrogen (~30 min).Blood sample extraction was carried out by combining 100 µl whole blood with 900 µl ice-cold extraction solvent .Urine sample extraction was carried out by adding 50 μl of urine into a SpinX filter and mixing it with 50 μL eluent B (10 mM ammonium acetate in 90 % acetonitrile). The filters were centrifugated at 3000 × Samples were handled during the whole extraction procedure at 4 °C, and sample analysis was started immediately after sample preparation without any intermediate storage. To ensure high-quality sample preparation, a quality control sample (QC sample) was prepared by pooling small equal aliquots from each sample for all approaches, to create a representative average of the entire set. This sample was treated and analyzed at regular intervals throughout the sequence.58, with the modification that deactivation additives were only added to the aqueous eluent and 5 µL were used as injection volume. Peak areas were extracted using Compound Discoverer 3.3 (Thermo Scientific).Polar metabolite profiling of whole blood and urine was performed using a Thermo Scientific Vanquish LC coupled to Thermo Q Exactive HF MS. An electrospray ionization interface was used as ionization source. Analysis was performed in negative and positive ionization mode. The UPLC was performed as described by Hsiao et al. 59. Peak areas were extracted using Compound Discoverer 3.3 (Thermo Scientific). The results from the two platforms were merged into one dataset. Compounds detected in both platforms were selected so the final data only contains the best representation of the compound.Semi-polar metabolite analysis of whole blood was carried out using a Thermo Scientific Vanquish LC coupled to Orbitrap Exploris 240 MS, Thermo Fisher Scientific. An electrospray ionization interface was used as ionization source. Analysis was performed in positive and negative ionization mode under polarity switching. The UPLC was performed using a the protocol described by Doneanu et al. For urine samples only polar extraction was performed. For blood samples the applied extraction method is indicated for each reported metabolite in Supplementary Data +, this is due to instability of the compound in the standard solutions. In the case of betaine and methionine, this is because those metabolites lacked a standard series for absolute quantification. Compounds with values available from less than 7 samples, were not included in downstream analyses.Identification of compounds for both methods was performed at four levels; Level 1: identification by retention times (compared against in-house authentic standards), accurate mass (with an accepted deviation of 3 ppm), and MS/MS spectra; Level 2a: identification by retention times (compared against in-house authentic standards), accurate mass (with an accepted deviation of 3 ppm). Level 2b: identification by accurate mass (with an accepted deviation of 3 ppm), and MS/MS spectra, Level 3: identification by accurate mass alone (with an accepted deviation of 3 ppm). All standard metabolites used here and the annotation level of the reported metabolites are indicated in Supplementary Data https://cran.r-project.org, version 4.2.2, R Foundation for Statistical Computing, Vienna, Austria) using the following packages; tidyverse version 1.3.1, readxl version 1.4.0, openxlsx version 4.2.5, lubridate version 1.8.0, cowplot version 1.1.1, ggpubr version 0.4.0, ggsignif version 0.6.3, rvg version 0.3.2, officer version 0.6.0 and effsize version 0.8.1.Data normality was tested by the Shapiro–Wilk test. The majority of variables in the dataset were normally distributed (Supplementary Data Further information on research design is available in the Peer Review FileDescription of Additional Supplementary FilesSupplementary Data 1Supplementary Data 2Supplementary Data 3Supplementary Data 4Supplementary Data 5Supplementary Data 6Supplementary Data 7Reporting SummarySource Data