Large-scale validation of skin prion seeding activity as a biomarker for diagnosis of prion diseases.

Definitive diagnosis of sporadic Creutzfeldt-Jakob disease (sCJD) relies on the examination of brain tissues for the pathological prion protein (PrPSc). Our previous study revealed that PrPSc-seeding activity (PrPSc-SA) is detectable in skin of sCJD patients by an ultrasensitive PrPSc seed amplification assay (PrPSc-SAA) known as real-time quaking-induced conversion (RT-QuIC). A total of 875 skin samples were collected from 2 cohorts (1 and 2) at autopsy from 2-3 body areas of 339 cases with neuropathologically confirmed prion diseases and non-sCJD controls. The skin samples were analyzed for PrPSc-SA by RT-QuIC assay. The results were compared with demographic information, clinical manifestations, cerebrospinal fluid (CSF) PrPSc-SA, other laboratory tests, subtypes of prion diseases defined by the methionine (M) or valine (V) polymorphism at residue 129 of PrP, PrPSc types (#1 or #2), and gene mutations in deceased patients. RT-QuIC assays of the cohort #1 by two independent laboratories gave 87.3% or 91.3% sensitivity and 94.7% or 100% specificity, respectively. The cohort #2 showed sensitivity of 89.4% and specificity of 95.5%. RT-QuIC of CSF available from 212 cases gave 89.7% sensitivity and 94.1% specificity. The sensitivity of skin RT-QuIC was subtype dependent, being highest in sCJDVV1-2 subtype, followed by VV2, MV1-2, MV1, MV2, MM1, MM1-2, MM2, and VV1. The skin area next to the ear gave highest sensitivity, followed by lower back and apex of the head. Although no difference in brain PrPSc-SA was detected between the cases with false negative and true positive skin RT-QuIC results, the disease duration was significantly longer with the false negatives [12.0 ± 13.3 (months, SD) vs. 6.5 ± 6.4, p < 0.001]. Our study validates skin PrPSc-SA as a biomarker for the detection of prion diseases, which is influenced by the PrPSc types, PRNP 129 polymorphisms, dermatome sampled, and disease duration.

Consistency evaluation of exercise target heart rate determined by resting heart rate and anaerobic threshold in chronic heart failure patients.

PURPOSE: To evaluate the consistency on the target heart rate for exercise determined by simple target heart rate (sTHR) based on resting heart rate (HRrest) and heart rate at anaerobic threshold (HRAT) in cardiopulmonary exercise test (CPET) for patients with chronic heart failure. METHODS: This is a retrospective cohort study, in which CHF patients who underwent CPET in Tongji Hospital Cardiac Rehabilitation Center Affiliated to Tongji University from March 2007 to December 2018 were enrolled. The clinical data of the patients from the electronic medical record system, HRrest and HRAT measured by CPET were collected. Patients were further divided into subgroups according to gender, age (<60 years group and ≥ 60 years group), with or without beta-blocker therapy and subgroup of heart failure (heart failure with reduced, mid-range and preserved ejection fraction). The sTHR (HRrest plus 10, 15, 20, 25 and 30 bpm) and HRAT were all calculated in each patient. Paired t-test was used for the difference between the two methods, correlation analysis was shown by pearson analysis and intraclass correlation coefficient (ICC) was calculated for consistency test. RESULTS: A total of 547 CHF patients were enrolled, including 447 males (81.7%), aged 63 (56,69) years, with BMI of 25.2 (23.5,26.4) kg/m2 and LVEF of 45.0 (36.0, 52.0) %. The target heart rate determined by HRAT method was (93.59 ± 13.95) bpm, and its counterpart determined by HRrest plus 20 bpm (HRrest+20) was (93.16 ± 7.69) bpm. There was no significant difference between the two methods (P>0.05). However, it was statistically different between HRrest plus 10, 15, 25, 30 bpm and HRAT respectively (P<0.001). And HRrest+20 was positively correlated with HRAT (r = 0.418, P<0.001). Therefore, HRrest+20 below was regarded as sTHR. The ICC of the consistency test between sTHR and HRAT was 0.523,95%CI 0.435-0.596 (P < 0.001) in all patients (n = 547). In patients with beta-blocker therapy (n = 464), the ICC of sTHR and HRAT consistency test was 0.534,95%CI 0.441-0.612, P < 0.001; The ICC of the consistency test between sTHR and HRAT of patients without beta-blocker therapy (n = 83) was 0.407,95%CI 0.083-0.616, P < 0.05. In the sinus rhythm group (n = 466), the ICC of sTHR and HRAT consistency test was 0.527,95%CI 0.433-0.606, P < 0.001; The ICC of the consistency test between sTHR and HRAT of atrial fibrillation patients in group (n = 81) was 0.482,95%CI 0.195-0.667, P < 0.05.The ICC of the consistency test between sTHR and HRAT was 0.501,95%CI 0.338-0.623 (P < 0.001) in patients under 60 years old (n = 195); The ICC of the consistency test between sTHR and HRAT in patients ≥60 years old (n = 352) was 0.533,95%CI 0.424-0.621, P < 0.001. In the male group (n = 447), the ICC of sTHR and HRAT consistency test was 0.577,95%CI 0.491-0.649, P < 0.001; The ICC of the consistency test between sTHR and HRAT of female patients in group (n = 100) was 0.344,95%CI 0.025-0.559, P < 0.05. The ICC of sTHR and HRAT consistency test in HFrEF group (n = 170) was 0.395,95%CI 0.181-0.553, P < 0.01; The ICC values of the consistency test between sTHR and HRAT was 0.543, 95%CI 0.405-0.649 (P < 0.001) in patients with HFmrEF (n = 222); In HFpEF group (n = 155), the ICC of sTHR and HRAT consistency test was 0.620,95%CI 0.478-0.723, P < 0.001. CONCLUSION: The exercise target heart rate calculated by HRrest is consistent with that determined by HRAT in patients with CHF. For primary hospitals without CPET, exercise prescription equivalent to AT intensity for patients with CHF can be determined by HRrest. However, the target heart rate calculated by HRrest can't replace that determined by HRAT in this patient cohort completely.

Stress and viral insults do not trigger E200K PrP conversion in human cerebral organoids.

Prion diseases are a group of rare, transmissible, and invariably fatal neurodegenerative diseases that affect both humans and animals. The cause of these diseases is misfolding of the prion protein into pathological isoforms called prions. Of all human prion diseases, 10-15% of cases are genetic and the E200K mutation, which causes familial Creutzfeldt-Jakob disease (CJD), is the most prevalent. For both sporadic and genetic disease, it remains uncertain as to how initial protein misfolding is triggered. Prior studies have linked protein misfolding with oxidative stress insults, deregulated interactions with cellular cofactors, and viral infections. Our previous work developed a cerebral organoid (CO) model using human induced pluripotent stem cells containing the E200K mutation. COs are three-dimensional human neural tissues that permit the study of host genetics and environmental factors that contribute to disease onset. Isogenically matched COs with and without the E200K mutation were used to investigate the propensity of E200K PrP to misfold following cellular insults associated with oxidative stress. Since viral infections have also been associated with oxidative stress and neurodegenerative diseases, we additionally investigated the influence of Herpes Simplex Type-1 virus (HSV1), a neurotropic virus that establishes life-long latent infection in its host, on E200K PrP misfolding. While COs proved to be highly infectable with HSV1, neither acute nor latent infection, or direct oxidative stress insult, resulted in evidence of E200K prion misfolding. We conclude that misfolding into seeding-active PrP species is not readily induced by oxidative stress or HSV1 in our organoid system.

Hereditary E200K mutation within the prion protein gene alters human iPSC derived cardiomyocyte function.

Cardiomyopathy is a co-morbidity of some prion diseases including genetic disease caused by mutations within the PrP gene (PRNP). Although the cellular prion protein (PrP) has been shown to protect against cardiotoxicity caused by oxidative stress, it is unclear if the cardiomyopathy is directly linked to PrP dysfunction. We differentiated cardiomyocyte cultures from donor human induced pluripotent stem cells and found a direct influence of the PRNP E200K mutation on cellular function. The PRNP E200K cardiomyocytes showed abnormal function evident in the irregularity of the rapid repolarization; a phenotype comparable with the dysfunction reported in Down Syndrome cardiomyocytes. PRNP E200K cardiomyocyte cultures also showed increased mitochondrial superoxide accompanied by increased mitochondrial membrane potential and dysfunction. To confirm that the changes were due to the E200K mutation, CRISPR-Cas9 engineering was used to correct the E200K carrier cells and insert the E200K mutation into control cells. The isotype matched cardiomyocytes showed that the lysine expressing allele does directly influence electrophysiology and mitochondrial function but some differences in severity were apparent between donor lines. Our results demonstrate that cardiomyopathy in hereditary prion disease may be directly linked to PrP dysfunction.

Neuronal excitatory-to-inhibitory balance is altered in cerebral organoid models of genetic neurological diseases.

The neuro-physiological properties of individuals with genetic pre-disposition to neurological disorders are largely unknown. Here we aimed to explore these properties using cerebral organoids (COs) derived from fibroblasts of individuals with confirmed genetic mutations including PRNPE200K, trisomy 21 (T21), and LRRK2G2019S, which are associated with Creutzfeldt Jakob disease, Down Syndrome, and Parkinson's disease. We utilized no known disease/healthy COs (HC) as normal function controls. At 3-4 and 6-10 months post-differentiation, COs with mutations showed no evidence of disease-related pathology. Electrophysiology assessment showed that all COs exhibited mature neuronal firing at 6-10 months old. At this age, we observed significant changes in the electrophysiology of the COs with disease-associated mutations (dCOs) as compared with the HC, including reduced neuronal network communication, slowing neuronal oscillations, and increased coupling of delta and theta phases to the amplitudes of gamma oscillations. Such changes were linked with the detection of hypersynchronous events like spike-and-wave discharges. These dysfunctions were associated with altered production and release of neurotransmitters, compromised activity of excitatory ionotropic receptors including receptors of kainate, AMPA, and NMDA, and changed levels and function of excitatory glutamatergic synapses and inhibitory GABAergic synapses. Neuronal properties that modulate GABAergic inhibition including the activity of Na-K-Cl cotransport 1 (NKCC1) in Cl- homeostasis and the levels of synaptic and extra-synaptic localization of GABA receptors (GABARs) were altered in the T21 COs only. The neurosteroid allopregnanolone, a positive modulator of GABARs, was downregulated in all the dCOs. Treatment with this neurosteroid significantly improved the neuronal communication in the dCOs, possibly through improving the GABAergic inhibition. Overall, without the manifestation of any disease-related pathology, the genetic mutations PRNPE200K, T21, and LRRK2G2019S significantly altered the neuronal network communication in dCOs by disrupting the excitatory-to-inhibitory balance.

New mechanism of partial duplication and deletion of chromosome 8: A case report.

BACKGROUND: During meiosis, the recombination of homologous chromosomes produces some new heritable mutations, which are the basis of biological evolution and diversity. However, when there is pericentric inversion of chromosomes, unbalanced gametes will be formed in the process of germ cell meiosis. CASE SUMMARY: A 23-year-old pregnant woman at 25 wk of gestation wanted to terminate her pregnancy due to fetal chromosomal abnormalities. She had no exposure to toxic or hazardous substances before and during pregnancy, no history of medication usage during pregnancy, and she underwent cystectomy of ovarian cysts in 2017. On the second day of the 16th week of gestation, non-invasive prenatal testing showed chromosome 8 copy number variation. Following genetic counseling, her pregnancy was terminated. CONCLUSION: Recombinant offspring chromosome is rarely seen when the inversion segment is shorter than one-third of the chromosome length. In terms of the mechanism of chromosome 8 duplication/deletion occurrence, attention should be paid to the production of unbalanced gametes by the pairing of homologous chromosome during meiosis, and the possibility of mitotic recombination exchange as well.

Generation of human chronic wasting disease in transgenic mice.

Chronic wasting disease (CWD) is a cervid prion disease caused by the accumulation of an infectious misfolded conformer (PrPSc) of cellular prion protein (PrPC). It has been spreading rapidly in North America and also found in Asia and Europe. Although bovine spongiform encephalopathy (i.e. mad cow disease) is the only animal prion disease known to be zoonotic, the transmissibility of CWD to humans remains uncertain. Here we report the generation of the first CWD-derived infectious human PrPSc by elk CWD PrPSc-seeded conversion of PrPC in normal human brain homogenates using in vitro protein misfolding cyclic amplification (PMCA). Western blotting with human PrP selective antibody confirmed that the PMCA-generated protease-resistant PrPSc was derived from the human PrPC substrate. Two lines of humanized transgenic mice expressing human PrP with either Val or Met at the polymorphic codon 129 developed clinical prion disease following intracerebral inoculation with the PMCA-generated CWD-derived human PrPSc. Diseased mice exhibited distinct PrPSc patterns and neuropathological changes in the brain. Our study, using PMCA and animal bioassays, provides the first evidence that CWD PrPSc can cross the species barrier to convert human PrPC into infectious PrPSc that can produce bona fide prion disease when inoculated into humanized transgenic mice.

Decrease in Skin Prion-Seeding Activity of Prion-Infected Mice Treated with a Compound Against Human and Animal Prions: a First Possible Biomarker for Prion Therapeutics.

Previous studies have revealed that the infectious scrapie isoform of prion protein (PrPSc) harbored in the skin tissue of patients or animals with prion diseases can be amplified and detected through the serial protein misfolding cyclic amplification (sPMCA) or real-time quaking-induced conversion (RT-QuIC) assays. These findings suggest that skin PrPSc-seeding activity may serve as a biomarker for the diagnosis of prion diseases; however, its utility as a biomarker for prion therapeutics remains largely unknown. Cellulose ethers (CEs, such as TC-5RW), widely used as food and pharmaceutical additives, have recently been shown to prolong the lifespan of prion-infected mice and hamsters. Here we report that in transgenic (Tg) mice expressing hamster cellular prion protein (PrPC) infected with the 263K prion, the prion-seeding activity becomes undetectable in the skin tissues of TC-5RW-treated Tg mice by both sPMCA and RT-QuIC assays, whereas such prion-seeding activity is readily detectable in the skin of untreated mice. Notably, TC-5RW exhibits an inhibitory effect on the in vitro amplification of PrPSc in both skin and brain tissues by sPMCA and RT-QuIC. Moreover, we reveal that TC-5RW is able to directly decrease protease-resistant PrPSc and inhibit the seeding activity of PrPSc from chronic wasting disease and various human prion diseases. Our results suggest that the level of prion-seeding activity in the skin may serve as a useful biomarker for assessing the therapeutic efficacy of compounds in a clinical trial of prion diseases and that TC-5RW may have the potential for the prevention/treatment of human prion diseases.

Further Characterization of Glycoform-Selective Prions of Variably Protease-Sensitive Prionopathy.

Prion is an infectious protein (PrPSc) that is derived from a cellular glycoprotein (PrPC) through a conformational transition and associated with a group of prion diseases in animals and humans. Characterization of proteinase K (PK)-resistant PrPSc by western blotting has been critical to diagnosis and understanding of prion diseases including Creutzfeldt-Jakob disease (CJD) and Gerstmann-Sträussler-Scheinker (GSS) disease in humans. However, formation as well as biochemical and biological properties of the glycoform-selective PrPSc in variably protease-sensitive prionopathy (VPSPr) remain poorly understood. Here we reveal that formation of the ladder-like PrPSc in VPSPr is a PK-dependent two-step process, which is enhanced by basic pH. Two sets of PrPSc fragments can be identified with antibodies directed against an intermediate or a C-terminal domain of the protein. Moreover, antibodies directed against specific PrP glycoforms reveal faster electrophoretic migrations of PrP fragments mono-glycosylated at residue 181 and 197 in VPSPr than those in sporadic CJD (sCJD). Finally, RT-QuIC assay indicates that PrPSc-seeding activity is lower and its lag time is longer in VPSPr than in sCJD. Our results suggest that the glycoform-selective PrPSc in VPSPr is associated with altered glycosylation, resulting in different PK-truncation and aggregation seeding activity compared to PrPSc in sCJD.

Characterization of Anchorless Human PrP With Q227X Stop Mutation Linked to Gerstmann-Sträussler-Scheinker Syndrome In Vivo and In Vitro.

Alteration in cellular prion protein (PrPC) localization on the cell surface through mediation of the glycosylphosphatidylinositol (GPI) anchor has been reported to dramatically affect the formation and infectivity of its pathological isoform (PrPSc). A patient with Gerstmann-Sträussler-Scheinker (GSS) syndrome was previously found to have a nonsense heterozygous PrP-Q227X mutation resulting in an anchorless PrP. However, the allelic origin of this anchorless PrPSc and cellular trafficking of PrPQ227X remain to be determined. Here, we show that PrPSc in the brain of this GSS patient is mainly composed of the mutant but not wild-type PrP (PrPWt), suggesting pathological PrPQ227X is incapable of recruiting PrPWt in vivo. This mutant anchorless protein, however, is able to recruit PrPWt from humanized transgenic mouse brain but not from autopsied human brain homogenates to produce a protease-resistant PrPSc-like form in vitro by protein misfolding cyclic amplification (PMCA). To further investigate the characteristics of this mutation, constructs expressing human PrPQ227X or PrPWt were transfected into neuroblastoma cells (M17). Fractionation of the M17 cells demonstrated that most PrPWt is recovered in the cell lysate fraction, while most of the mutant PrPQ227X is recovered in the medium fraction, consistent with the results obtained by immunofluorescence microscopy. Two-dimensional gel-electrophoresis and Western blotting showed that cellular PrPQ227X spots clustered at molecular weights of 22-25 kDa with an isoelectric point (pI) of 3.5-5.5, whereas protein spots from the medium are at 18-26 kDa with a pI of 7-10. Our findings suggest that the role of GPI anchor in prion propagation between the anchorless mutant PrP and wild-type PrP relies on the cellular distribution of the protein.

Skin α-Synuclein Aggregation Seeding Activity as a Novel Biomarker for Parkinson Disease.

IMPORTANCE: Deposition of the pathological α-synuclein (αSynP) in the brain is the hallmark of synucleinopathies, including Parkinson disease (PD), Lewy body dementia (LBD), and multiple system atrophy (MSA). Whether real-time quaking-induced conversion (RT-QuIC) and protein misfolding cyclic amplification (PMCA) assays can sensitively detect skin biomarkers for PD and non-PD synucleinopathies remains unknown. OBJECTIVE: To develop sensitive and specific skin biomarkers for antemortem diagnosis of PD and other synucleinopathies. DESIGN, SETTING, AND PARTICIPANTS: This retrospective and prospective diagnostic study evaluated autopsy and biopsy skin samples from neuropathologically and clinically diagnosed patients with PD and controls without PD. Autopsy skin samples were obtained at 3 medical centers from August 2016 to September 2019, and biopsy samples were collected from 3 institutions from August 2018 to November 2019. Based on neuropathological and clinical diagnoses, 57 cadavers with synucleinopathies and 73 cadavers with nonsynucleinopathies as well as 20 living patients with PD and 21 living controls without PD were included. Specifically, cadavers and participants had PD, LBD, MSA, Alzheimer disease, progressive supranuclear palsy, or corticobasal degeneration or were nonneurodegenerative controls (NNCs). A total of 8 approached biopsy participants either refused to participate in or were excluded from this study due to uncertain clinical diagnosis. Data were analyzed from September 2019 to April 2020. MAIN OUTCOMES AND MEASURES: Skin αSynP seeding activity was analyzed by RT-QuIC and PMCA assays. RESULTS: A total of 160 autopsied skin specimens from 140 cadavers (85 male cadavers [60.7%]; mean [SD] age at death, 76.8 [10.1] years) and 41 antemortem skin biopsies (27 male participants [66%]; mean [SD] age at time of biopsy, 65.3 [9.2] years) were analyzed. RT-QuIC analysis of αSynP seeding activity in autopsy abdominal skin samples from 47 PD cadavers and 43 NNCs revealed 94% sensitivity (95% CI, 85-99) and 98% specificity (95% CI, 89-100). As groups, RT-QuIC also yielded 93% sensitivity (95% CI, 85-97) and 93% specificity (95% CI, 83-97) among 57 cadavers with synucleinopathies (PD, LBD, and MSA) and 73 cadavers without synucleinopathies (Alzheimer disease, progressive supranuclear palsy, corticobasal degeneration, and NNCs). PMCA showed 82% sensitivity (95% CI, 76-88) and 96% specificity (95% CI, 85-100) with autopsy abdominal skin samples from PD cadavers. From posterior cervical and leg skin biopsy tissues from patients with PD and controls without PD, the sensitivity and specificity were 95% (95% CI, 77-100) and 100% (95% CI, 84-100), respectively, for RT-QuIC and 80% (95% CI, 49-96) and 90% (95% CI, 60-100) for PMCA. CONCLUSIONS AND RELEVANCE: This study provides proof-of-concept that skin αSynP seeding activity may serve as a novel biomarker for antemortem diagnoses of PD and other synucleinopathies.

Integrated epigenetic biomarkers in circulating cell-free DNA as a robust classifier for pancreatic cancer.

BACKGROUND: The high lethal rate of pancreatic cancer is partly due to a lack of efficient biomarkers for screening and early diagnosis. We attempted to develop effective and noninvasive methods using 5-methylcytosine (5mC) and 5-hydroxymethylcytosine (5hmC) markers from circulating cell-free DNA (cfDNA) for the detection of pancreatic ductal adenocarcinoma (PDAC). RESULTS: A 24-feature 5mC model that can accurately discriminate PDAC from healthy controls (area under the curve (AUC) = 0.977, sensitivity = 0.824, specificity = 1) and a 5hmC prediction model with 27 features demonstrated excellent detection power in two distinct validation sets (AUC = 0.992 and 0.960, sensitivity = 0.786 and 0.857, specificity = 1 and 0.993). The 51-feature model combining 5mC and 5hmC markers outperformed both of the individual models, with an AUC of 0.997 (sensitivity = 0.938, specificity = 0.955) and particularly an improvement in the prediction sensitivity of PDAC. In addition, the weighted diagnosis score (wd-score) calculated with the 5hmC model can distinguish stage I patients from stage II-IV patients. CONCLUSIONS: Both 5mC and 5hmC biomarkers in cfDNA are effective in PDAC detection, and the 5mC-5hmC integrated model significantly improve the detection sensitivity.

Pathogenic Prion Protein Isoforms Are Not Present in Cerebral Organoids Generated from Asymptomatic Donors Carrying the E200K Mutation Associated with Familial Prion Disease.

Cerebral organoids (COs) are a self-organizing three-dimensional brain tissue mimicking the human cerebral cortex. COs are a promising new system for modelling pathological features of neurological disorders, including prion diseases. COs expressing normal prion protein (PrPC) are susceptible to prion infection when exposed to the disease isoforms of PrP (PrPD). This causes the COs to develop aspects of prion disease pathology considered hallmarks of disease, including the production of detergent-insoluble, protease-resistant misfolded PrPD species capable of seeding the production of more misfolded species. To determine whether COs can model aspects of familial prion diseases, we produced COs from donor fibroblasts carrying the E200K mutation, the most common cause of human familial prion disease. The mature E200K COs were assessed for the hallmarks of prion disease. We found that up to 12 months post-differentiation, E200K COs harbored no PrPD as confirmed by the absence of detergent-insoluble, protease-resistant, and seeding-active PrP species. Our results suggest that the presence of the E200K mutation within the prion gene is insufficient to cause disease in neuronal tissue. Therefore, other factors, such as further genetic modifiers or aging processes, may influence the onset of misfolding.

Oral microbiome and pancreatic cancer.

BACKGROUND: Microbiota profiles differ between patients with pancreatic cancer and healthy people, and understanding these differences may help in early detection of pancreatic cancer. Saliva sampling is an easy and cost-effective way to determine microbiota profiles compared to fecal and tissue sample collection. AIM: To investigate the saliva microbiome distribution in patients with pancreatic adenocarcinoma (PDAC) and the role of oral microbiota profiles in detection and risk prediction of pancreatic cancer. METHODS: We conducted a prospective study of patients with pancreatic cancer (n = 41) and healthy individuals (n = 69). Bacterial taxa were identified by 16S ribosomal ribonucleic acid gene sequencing, and a linear discriminant analysis effect size algorithm was used to identify differences in taxa. Operational taxonomic unit values of all selected taxa were converted into a normalized Z-score, and logistic regressions were used to calculate risk prediction of pancreatic cancer. RESULTS: Compared with the healthy control group, carriage of Streptococcus and Leptotrichina (z-score) was associated with a higher risk of PDAC [odds ratio (OR) = 5.344, 95% confidence interval (CI): 1.282-22.282, P = 0.021 and OR = 6.886, 95%CI: 1.423-33.337, P = 0.016, respectively]. Veillonella and Neisseria (z-score) were considered a protective microbe that decreased the risk of PDAC (OR = 0.187, 95%CI: 0.055-0.631, P = 0.007 and OR = 0.309, 95%CI: 0.100-0.952, P = 0.041, respectively). Among the patients with PDAC, patients reporting bloating have a higher abundance of Porphyromonas (P = 0.039), Fusobacterium (P = 0.024), and Alloprevotella (P = 0.041); while patients reporting jaundice had a higher amount of Prevotella (P = 0.008); patients reporting dark brown urine had a higher amount of Veillonella (P = 0.035). Patients reporting diarrhea had a lower amount of Neisseria and Campylobacter (P = 0.024 and P = 0.034), and patients reporting vomiting had decreased Alloprevotella (P = 0.036). CONCLUSION: Saliva microbiome was able to distinguish patients with pancreatic cancer and healthy individuals. Leptotrichia may be specific for patients living in Sichuan Province, southwest China. Symptomatic patients had different bacteria profiles than asymptomatic patients. Combined symptom and microbiome evaluation may help in the early detection of pancreatic cancer.

[Psychometric Evaluation of the Union Physio-Psycho-Social Assessment Questionnaire].

Objective To validate the Union Physio-Psycho-Social Assessment Questionnaire(UPPSAQ-70)and test its validity and reliability.Methods From April,2013 to July,2018,patients were asked to finish the computer evaluation of UPPSAQ-70 and Symptom Checklist 90(SCL-90)in Peking Union Medical College Hospital(PUMCH).Confirmatory factor analysis(CFA)was conducted on the SPSS 17.0,and the number of fixed factors was 8 factors and 3 factors.Amos 23.0 was used to verify the original 8-factor model,8-factor revision model,3-factor model,3-factor revision model,and single-factor model.Each factor of SCL-90 was used as the calibration standard to calculate the correlation coefficient between factors.The retest reliability was tested by the outpatients in PUMCH in July,2018.Results Exploratory factor analysis indicated that the 8-factor revised model included:depression,anxiety and fatigue,sleep,physical discomfort,sexual function,happiness and satisfaction,hypochondria,and social anxiety.The 3 factors revised model included that:psychological,physiological and social dimension.Confirmatory factor analysis indicated that the 8-factor modified model was superior to the 3-factor model and the single-factor model: χ 2=10 410.4,df=1862,RMSEA=0.07,CFI=0.753,and NFI=0.715.With SCL-90 as the standard criteria,except the low correlation coefficient between emotional scale and depression(r=0.600)and anxiety(r=0.520),the correlation coefficients of other symptoms were below 0.5.The chronbach's α between each factor and total score of UPPSAQ-70 was between 0.823 and 0.904,and the Chronbach's α coefficient of the whole scale was between 0.954 and 0.956 after each item was deleted.The retest reliability of the scale of 32 participants Chronbach's α was 0.847.Each item of the scale measured between one week was significantly correlated(P<0.05). Conclusion UPPSAQ-70 is a good scale for evaluating overall health status and is especially feasible in general hospitals.

Publisher Correction: Early preclinical detection of prions in the skin of prion-infected animals.

The original version of this Article contained errors in the author affiliations. Affiliation 2 incorrectly read 'Department of Neurology, The First Hospital of Jilin University, Changchun 130021 Jilin Province, China.'Affiliation 5 incorrectly read 'Department of Otolaryngology, The First Affiliated Hospital of Xi'an Jiaotong University, Xi'an 710061 Shanxi Province, China'Affiliation 9 incorrectly read 'State Key Laboratory for Infectious Disease Prevention and Control, National Institute for Viral Disease Control and Prevention, Chinese Center for Disease Control and Prevention, Beijing 102206, China.'This has now been corrected in both the PDF and HTML versions of the Article.

Correction to: In Vitro Seeding Activity of Glycoform-Deficient Prions from Variably Protease-Sensitive Prionopathy and Familial CJD Associated with PrPV180I Mutation.

The original version of this article unfortunately contained a mistake. The email address Dr. Wen-Quan Zou, one of the corresponding authors should be written as "wxz6@case.edu" instead of "wxz@case.edu".

Early preclinical detection of prions in the skin of prion-infected animals.

A definitive pre-mortem diagnosis of prion disease depends on brain biopsy for prion detection currently and no validated alternative preclinical diagnostic tests have been reported to date. To determine the feasibility of using skin for preclinical diagnosis, here we report ultrasensitive serial protein misfolding cyclic amplification (sPMCA) and real-time quaking-induced conversion (RT-QuIC) assays of skin samples from hamsters and humanized transgenic mice (Tg40h) at different time points after intracerebral inoculation with 263K and sCJDMM1 prions, respectively. sPMCA detects skin PrPSc as early as 2 weeks post inoculation (wpi) in hamsters and 4 wpi in Tg40h mice; RT-QuIC assay reveals earliest skin prion-seeding activity at 3 wpi in hamsters and 20 wpi in Tg40h mice. Unlike 263K-inoculated animals, mock-inoculated animals show detectable skin/brain PrPSc only after long cohabitation periods with scrapie-infected animals. Our study provides the proof-of-concept evidence that skin prions could be a biomarker for preclinical diagnosis of prion disease.

In Vitro Seeding Activity of Glycoform-Deficient Prions from Variably Protease-Sensitive Prionopathy and Familial CJD Associated with PrPV180I Mutation.

Both sporadic variably protease-sensitive prionopathy (VPSPr) and familial Creutzfeldt-Jakob disease linked to the prion protein (PrP) V180I mutation (fCJDV180I) have been found to share a unique pathological prion protein (PrPSc) that lacks the protease-resistant PrPSc glycosylated at residue 181 because two of four PrP glycoforms are apparently not converted into the PrPSc from their cellular PrP (PrPC). To investigate the seeding activity of these unique PrPSc molecules, we conducted in vitro prion conversion experiments using serial protein misfolding cyclic amplification (sPMCA) and real-time quaking-induced conversion (RT-QuIC) assays with different PrPC substrates. We observed that the seeding of PrPSc from VPSPr or fCJDV180I in the sPMCA reaction containing normal human or humanized transgenic (Tg) mouse brain homogenates generated PrPSc molecules that unexpectedly exhibited a dominant diglycosylated PrP isoform along with PrP monoglycosylated at residue 181. The efficiency of PrPSc amplification was significantly higher in non-CJDMM than in non-CJDVV human brain homogenate, whereas it was higher in normal TgVV than in TgMM mouse brain homogenate. PrPC from the mixture of normal TgMM and Tg mouse brain expressing PrPV180I mutation (Tg180) but not TgV180I alone was converted into PrPSc by seeding with the VPSPr or fCJDV180I. The RT-QuIC seeding activity of PrPSc from VPSPr and fCJDV180I was significantly lower than that of sCJD. Our results suggest that the formation of glycoform-selective prions may be associated with an unidentified factor in the affected brain and the glycoform-deficiency of PrPSc does not affect the glycoforms of in vitro newly amplified PrPSc.