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Background and ObjectivesReal-time quaking-induced conversion (RT-QuIC) assay detects misfolded α-synuclein (AS) in the skin and CSF of patients with the synucleinopathies Parkinson disease and dementia with Lewy bodies. Isolated REM sleep behavior disorder (IRBD) constitutes the prodromal stage of these synucleinopathies. We aimed to compare the ability of RT-QuIC to identify AS in the skin and CSF of patients with IRBD.MethodsThis was a cross-sectional study where consecutive patients with polysomnographic-confirmed IRBD and age-matched controls without RBD underwent skin biopsy and lumbar puncture the same day. Three-millimeter skin punch biopsies were obtained bilaterally in the cervical region from dorsal C7 and C8 dermatomes and in distal legs. RT-QuIC assessed AS in these 6 skin sites and the CSF.ResultsWe recruited 91 patients with IRBD and 41 controls. In the skin, sensitivity to detect AS was 76.9% (95% CI 66.9–85.1), specificity 97.6% (95% CI 87.1–99.9) positive predictive value 98.6% (95% CI 91.0–99.8), negative predictive value 65.6% (95% CI 56.6–73.6), and accuracy 83.3% (95% CI 75.9–89.3). In the CSF, the sensitivity was 75.0% (95% CI 64.6–83.6), the specificity was 97.5% (95% CI 86.8–99.9), the positive predictive value was 98.5% (95% CI 90.5–99.8), the negative predictive value was 63.9% (95% CI 55.2–71.9), and the accuracy was 82.0% (95% CI 74.3–88.3). Results in the skin and CSF samples showed 99.2% agreement. Compared with negative patients, RT-QuIC AS-positive patients had a higher likelihood ratio of prodromal Parkinson disease (p< 0.001) and showed more frequently hyposmia (p< 0.001), dopamine transporter imaging single-photon emission CT deficit (p= 0.002), and orthostatic hypotension (p= 0.014). No severe or moderate adverse effects were reported. There was no difference between the percentage of participants reporting mild adverse events secondary to skin biopsy or lumbar puncture (9.1% vs 17.2%;p= 0.053). One hundred and ten (83%) and 104 (80%) participants, respectively, stated they would accept to undergo skin biopsy and lumbar puncture again for research purposes.DiscussionOur study in IRBD shows that (1) RT-QuIC detects AS in the skin and CSF with similar high sensitivity, specificity, and agreement, (2) AS RT-QuIC positivity is associated with supportive features and biomarkers of synucleinopathy, and (3) skin punch biopsy and lumbar puncture have comparable mild adverse effects, tolerance, and acceptance. RT-QuIC in the skin or CSF might represent a patient selection strategy for future neuroprotective trials targeting AS in IRBD.Classification of EvidenceThis study provides Class III evidence that RT-QuIC–detected AS in the skin and CSF distinguishes patients with IRBD from controls.

BackgroundThe introduction of the prion Real-Time Quaking-Induced Conversion assay (RT-QuIC) has led to a revision of the diagnostic criteria for sporadic Creutzfeldt-Jakob disease (sCJD).Validation studies are needed for the amended criteria, especially for their diagnostic value in the clinical setting.MethodsWe studied 1250 patients with suspected CJD referred for diagnosis to two Italian reference centres between 2010 and 2020. Focusing on the first diagnostic assessment, we compared the diagnostic value of the old and the amended criteria and that of different combinations of clinical variables and biomarker results.ResultsThe studied cohort comprised 850 participants with CJD (297 definite sCJD, 151 genetic CJD, 402 probable sCJD) and 400 with non-CJD (61 with neuropathology). At first clinical evaluation, the sensitivity of the old criteria (76.8%) was significantly lower than that of the amended criteria (97.8%) in the definite CJD cohort with no difference between definite and probable sCJD cases. Specificity was ~94% for both criteria against the non-CJD cohort (82.0% against definite non-CJD group). Cerebrospinal fluid (CSF) RT-QuIC was highly sensitive (93.9%) and fully specific against definite non-CJD patients. Limiting the criteria to a positive RT-QuIC or/and the combination of a clinical course compatible with possible CJD with a positive MRI (Q-CM criteria) provided higher diagnostic accuracy than both the old and amended criteria, overcoming the suboptimal specificity of ancillary test results (ie, CSF protein 14-3-3).ConclusionsCSF RT-QuIC is highly sensitive and specific for diagnosing CJD in vitam. The Q-CM criteria provide a high diagnostic value for CJD.

The current classification of sporadic Creutzfeldt–Jakob disease identifies six major subtypes mainly defined by the combination of the genotype at polymorphic codon 129 (methionine/M or valine/V) of the prion protein gene and the type (1 or 2) of misfolded prion protein accumulating in the brain (e.g. MM1, MM2, MV1, MV2, etc.). Here, we systematically characterized the clinical and histo-molecular features associated with the third prevalent subtype, the MV2 subtype with kuru plaques (MV2K), in the most extensive series collected to date. We evaluated neurological histories, cerebrospinal biomarkers, brain MRI and EEG results in 126 patients. The histo-molecular assessment included misfolded prion protein typing, standard histologic staining and immunohistochemistry for prion protein in several brain areas. We also investigated the prevalence and topographic extent of coexisting MV2-cortical features, the number of cerebellar kuru plaques and their effect on clinical phenotype. Systematic regional typing revealed a western blot profile of misfolded prion protein comprising a doublet of 19 and 20 kDa unglycosylated fragments, with the former more prominent in neocortices and the latter in the deep grey nuclei. The 20/19 kDa fragment ratio positively correlated with the number of cerebellar kuru plaques. The mean disease duration was exceedingly longer than in the typical MM1 subtype (18.0 versus 3.4 months). Disease duration correlated positively with the severity of pathologic change and the number of cerebellar kuru plaques. At the onset and early stages, patients manifested prominent, often mixed, cerebellar symptoms and memory loss, variably associated with behavioural/psychiatric and sleep disturbances. The cerebrospinal fluid prion real-time quaking-induced conversion assay was positive in 97.3% of cases, while 14-3-3 protein and total-tau positive tests were 52.6 and 75.9%. Brain diffusion-weighted MRI showed hyperintensity of the striatum, cerebral cortex and thalamus in 81.4, 49.3 and 33.8% of cases, and a typical profile in 92.2%. Mixed histotypes (MV2K + MV2-cortical) showed an abnormal cortical signal more frequently than the pure MV2K (64.7 versus 16.7%, P = 0.007). EEG revealed periodic sharp-wave complexes in only 8.7% of participants. These results further establish MV2K as the most common ‘atypical’ subtype of sporadic Creutzfeldt–Jakob disease, showing a clinical course that often challenges the early diagnosis. The plaque-type aggregation of the misfolded prion protein accounts for most of the atypical clinical features. Nonetheless, our data strongly suggest that the consistent use of the real-time quaking-induced conversion assay and brain diffusion-weighted MRI allows an accurate early clinical diagnosis in most patients.

Background and ObjectivesFor early diagnosis and disease monitoring of neurodegenerative diseases (NDs), reliable blood biomarkers are needed. Elevated levels of neurofilament light chain protein (NfL), an axonal damage marker, have been described across different NDs, with highest values in prion diseases and amyotrophic lateral sclerosis (ALS). Synaptic degeneration is a common early feature in most NDs and seems to precede neuronal degeneration in prion disease. However, synaptic markers in blood are still missing. Here, we investigated whether the brain-specific protein β-synuclein might be a suitable blood biomarker for early diagnosis and evaluation of synaptic integrity in prion disease.MethodsWe analyzed blood β-synuclein with a newly established digital ELISA and NfL with a single-molecule array in samples obtained from human participants and prion and ALS animal models. Furthermore, β-synuclein was investigated in brain tissue of individuals with Creutzfeldt-Jakob disease (CJD) and controls.ResultsWe investigated 308 patients, including 129 cases with prion disease, 8 presymptomatic PRNP variation carriers, 60 with ALS, 68 with other ND, and 43 control patients. In CJD symptomatic cases, β-synuclein and NfL were markedly increased compared to all other diagnostic groups (p < 0.001). In the large majority of presymptomatic PRNP variation carriers, β-synuclein and NfL levels were within normal ranges. In prion disease animal models, β-synuclein and NfL displayed normal levels in the presymptomatic phase with a sudden elevation at disease onset and a plateau in the symptomatic phase. In contrast to NfL, β-synuclein was not elevated in either symptomatic patients with ALS or an ALS animal model. In the discrimination between prion disease and all other groups, β-synuclein (area under the curve 0.97, 95% CI 0.94–0.99, p < 0.001) was superior to NfL (area under the curve 0.91, 95% CI 0.88–0.94, p < 0.001). In addition, brain tissue β-synuclein showed significantly reduced levels in patients with CJD compared to control patients (p < 0.001).DiscussionBlood β-synuclein was significantly elevated in patients with CJD, reflecting ongoing synaptic damage, and showed good discriminative characteristics. We therefore propose it as a candidate blood marker for early diagnosis and monitoring of synaptic integrity in prion disease.Classification of EvidenceThis study provides Class III evidence that serum β-synuclein concentration accurately distinguishes patients with symptomatic CJD from controls.

Background: Evidence suggests that skin represents a suitable matrix for demonstrating α-synuclein oligomers as a diagnostic biomarker for Lewy body disease. Objective: The objective of this study was to evaluate the diagnostic performance of skin α-syn real-time quaking-induced conversion assay in patients with Lewy body disease. Methods: We analyzed skin punches taken in vitam (n=69) or postmortem (n=49) from patients with PD, dementia with Lew bodies (DLB), incidental Lewy body pathology, and neurological controls. Seventy-nine patients underwent both CSF and skin α-synuclein real-time quaking-induced conversion assay. Results: Overall, the skin α-synuclein real-time quaking-induced conversion assay distinguished Lewy body disease patients with 94.1% accuracy (sensitivity, 89.2%; specificity, 96.3%). Assay sensitivity reached 94.1% in the 17 Lewy body disease patients analyzed in the cervical region. In patients with both CSF and skin samples, the 2 real-time quaking-induced conversion assay protocols yielded similar diagnostic accuracy (skin, 97.5%; CSF, 98.7%). Conclusion: Skin punch biopsies might represent a valid and convenient alternative to CSF analysis to demonstrate Lew body-related α-synuclein deposition in patients with Lewy body disease. © 2021 The Authors. Movement Disorders published by Wiley Periodicals LLC on behalf of International Parkinson and Movement Disorder Society.

Background Increasing evidence supports the use of plasma biomarkers of neurodegeneration and neuroinflammation to screen and diagnose patients with dementia. However, confirmatory studies are required to demonstrate their usefulness in the clinical setting. Methods We evaluated plasma and cerebrospinal fluid (CSF) samples from consecutive patients with frontotemporal dementia (FTD) (n = 59), progressive supranuclear palsy (PSP) (n = 31), corticobasal syndrome (CBS) (n = 29), dementia with Lewy bodies (DLB) (n = 49), Alzheimer disease (AD) (n = 97), and suspected non-AD physiopathology (n = 51), as well as plasma samples from 60 healthy controls (HC). We measured neurofilament light chain (NfL), phospho-tau181 (p-tau181), and glial fibrillary acid protein (GFAP) using Simoa (all plasma biomarkers and CSF GFAP), CLEIA (CSF p-tau181), and ELISA (CSF NfL) assays. Additionally, we stratified patients according to the A/T/N classification scheme and the CSF α-synuclein real-time quaking-induced conversion assay (RT-QuIC) results. Results We found good correlations between CSF and plasma biomarkers for NfL (rho = 0.668, p < 0.001) and p-tau181 (rho = 0.619, p < 0.001). Plasma NfL was significantly higher in disease groups than in HC and showed a greater increase in FTD than in AD [44.9 (28.1–68.6) vs. 21.9 (17.0–27.9) pg/ml, p < 0.001]. Conversely, plasma p-tau181 and GFAP levels were significantly higher in AD than in FTD [3.2 (2.4–4.3) vs. 1.1 (0.7–1.6) pg/ml, p < 0.001; 404.7 (279.7–503.0) vs. 198.2 (143.9–316.8) pg/ml, p < 0.001]. GFAP also allowed discriminating disease groups from HC. In the distinction between FTD and AD, plasma p-tau181 showed better accuracy (AUC 0.964) than NfL (AUC 0.791) and GFAP (AUC 0.818). In DLB and CBS, CSF amyloid positive (A+) subjects had higher plasma p-tau181 and GFAP levels than A− individuals. CSF RT-QuIC showed positive α-synuclein seeding activity in 96% DLB and 15% AD patients with no differences in plasma biomarker levels in those stratified by RT-QuIC result. Conclusions In a single-center clinical cohort, we confirm the high diagnostic value of plasma p-tau181 for distinguishing FTD from AD and plasma NfL for discriminating degenerative dementias from HC. Plasma GFAP alone differentiates AD from FTD and neurodegenerative dementias from HC but with lower accuracy than p-tau181 and NfL. In CBS and DLB, plasma p-tau181 and GFAP levels are significantly influenced by beta-amyloid pathology.

The clinical diagnosis of synucleinopathies, including Parkinson’s disease (PD), dementia with Lewy bodies (DLB), and multiple system atrophy (MSA), is challenging, especially at an early disease stage, due to the heterogeneous and often non-specific clinical manifestations. The discovery of reliable specific markers for synucleinopathies would consequently be of great aid to the diagnosis and management of these disorders. Real-Time Quaking-Induced Conversion (RT-QuIC) is an ultrasensitive technique that has been previously used to detect self-templating amyloidogenic proteins in the cerebrospinal fluid (CSF) and other biospecimens in prion disease and synucleinopathies. Using a wild-type recombinant α-synuclein as a substrate, we applied RT-QuIC to a large cohort of 439 CSF samples from clinically well-characterized, or post-mortem verified patients with parkinsonism or dementia. Of significance, we also studied patients with isolated REM sleep behavior disorder (iRBD) (n = 18) and pure autonomic failure (PAF) (n = 28), representing clinical syndromes that are often caused by a synucleinopathy, and may precede the appearance of parkinsonism or cognitive decline. The results show that our RT-QuIC assay can accurately detect α-synuclein seeding activity across the spectrum of Lewy Body (LB)-related disorders (LBD), including DLB, PD, iRBD, and PAF, with an overall sensitivity of 95.3%. In contrast, all but two patients with MSA showed no α-synuclein seeding activity in the applied experimental setting. The analysis of the fluorescence response reflecting the amount of α-synuclein seeds revealed no significant differences between the clinical syndromes associated with LB pathology. Finally, the assay demonstrated 98% specificity in a neuropathological cohort of 101 cases lacking LB pathology. In conclusion, α-synuclein RT-QuIC provides an accurate marker of synucleinopathies linked to LB pathology and may have a pivotal role in the early discrimination and management of affected patients. The finding of no α-synuclein seeding activity in MSA seems to support the current view that MSA and LBD are associated with different conformational strains of α-synuclein. Electronic supplementary material The online version of this article (10.1007/s00401-020-02160-8) contains supplementary material, which is available to authorized users.

Prion real‐time quaking‐induced conversion (RT‐QuIC) is an ultrasensitive assay detecting pathological aggregates of misfolded prion protein in biospecimens. We studied 71 punch biopsy skin samples of 35 patients with Creutzfeldt–Jakob disease (CJD), including five assessed in vitam. The results confirmed the high value of skin prion RT‐QuIC for CJD diagnosis (89% sensitivity and 100% specificity) and support its use in clinical practice. Preliminary data based on a limited number of cases suggest that prion‐seeding activity in the skin varies according to the prion strain, being higher in sporadic CJD subtypes linked to the V2 strain (VV2 and MV2K) than in typical CJDMM1.

Variably protease-sensitive prionopathy is an exceedingly rare, likely underestimated, sporadic prion disease that is characterized by heterogeneous and often non-specific clinical and pathological features posing diagnostic challenges. We report the results of a comprehensive analysis of three emblematic cases carrying different genotypes at the methionine (M)/valine (V) polymorphic codon 129 in the prion protein gene (

ObjectiveTo investigate whether the CSF α-synuclein (α-syn) real-time quaking-induced conversion (RT-QuIC) assay accurately identifies patients with mild cognitive impairment (MCI) due to probable Lewy body (LB) disease.MethodsWe applied α-syn RT-QuIC to 289 CSF samples obtained from 2 independent cohorts, including 81 patients with probable MCI-LB (age 70.7 ± 6.6 years, 13.6% female, Mini-Mental State Examination [MMSE] score 26.1 ± 2.4), 120 with probable MCI due to Alzheimer disease (AD) (age 68.6 ± 7.4 years, 45.8% female, MMSE score 25.5 ± 2.8), and 30 with unspecified MCI (age 65.4 ± 9.3 years, 30.0% female, MMSE score 27.0 ± 3.0). Fifty-eight individuals with no cognitive decline or evidence of neurodegenerative disease and 121 individuals lacking brain α-syn deposits at the neuropathologic examination were used as controls.ResultsRT-QuIC identified patients with MCI-LB against cognitively unimpaired controls with 95% sensitivity, 97% specificity, and 96% accuracy and showed 98% specificity in neuropathologic controls. The accuracy of the test for MCI-LB was consistent between the 2 cohorts (97.3% vs 93.7%). Thirteen percent of patients with MCI-AD also had a positive test; of note, 44% of them developed 1 core or supportive clinical feature of dementia with Lewy bodies (DLB) at follow-up, suggesting an underlying LB copathology.ConclusionsThese findings indicate that CSF α-syn RT-QuIC is a robust biomarker for prodromal DLB. Further studies are needed to fully explore the added value of the assay to the current research criteria for MCI-LB.Classification of EvidenceThis study provides Class III evidence that CSF α-syn RT-QuIC accurately identifies patients with MCI-LB.

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The current classification of sporadic Creutzfeldt–Jakob disease (sCJD) includes six major clinicopathological subtypes defined by the physicochemical properties of the protease-resistant core of the pathologic prion protein (PrPSc), defining two major PrPSc types (i.e., 1 and 2), and the methionine (M)/valine (V) polymorphic codon 129 of the prion protein gene (PRNP). How these sCJD subtypes relate to the well-documented phenotypic heterogeneity of genetic CJD (gCJD) is not fully understood. We analyzed molecular and phenotypic features in 208 individuals affected by gCJD, carrying 17 different mutations, and compared them with those of a large series of sCJD cases. We identified six major groups of gCJD based on the combination PrPSc type and codon 129 genotype on PRNP mutated allele, each showing distinctive histopathological characteristics, irrespectively of the PRNP associated mutation. Five gCJD groups, named M1, M2C, M2T, V1, and V2, largely reproduced those previously described in sCJD subtypes. The sixth group shared phenotypic traits with the V2 group and was only detected in patients carrying the E200K-129M haplotype in association with a PrPSc type of intermediate size (“i”) between type 1 and type 2. Additional mutation-specific effects involved the pattern of PrP deposition (e.g., a “thickened” synaptic pattern in E200K carriers, cerebellar “stripe-like linear granular deposits” in those with insertion mutations, and intraneuronal globular dots in E200K-V2 or -M”i”). A few isolated cases linked to rare PRNP haplotypes (e.g., T183A-129M), showed atypical phenotypic features, which prevented their classification into the six major groups. The phenotypic variability of gCJD is mostly consistent with that previously found in sCJD. As in sCJD, the codon 129 genotype and physicochemical properties of PrPSc significantly correlated with the phenotypic variability of gCJD. The most common mutations linked to CJD appear to have a variable and overall less significant effect on the disease phenotype, but they significantly influence disease susceptibility often in a strain-specific manner. The criteria currently used for sCJD subtypes can be expanded and adapted to gCJD to provide an updated classification of the disease with a molecular basis.

Introduction Surrogate cerebrospinal fluid (CSF) biomarkers of neurodegeneration still have a central role in the first‐line screening of patients with suspected Creutzfeldt‐Jakob disease (CJD). Recently, CSF α‐synuclein, a marker of synaptic damage, showed a close to optimal performance in distinguishing between CJD and other neurodegenerative dementias. Methods We evaluated the diagnostic value of CSF α‐synuclein in patients with prion disease, non‐prion rapidly progressive dementias, and non‐neurodegenerative controls. Additionally, we studied its distribution across the different prion disease subtypes and evaluated its association with survival. Results CSF α‐synuclein levels were significantly higher in patients with prion disease than in the other groups but showed a lower diagnostic value than CSF total tau or 14‐3‐3. Moreover, CSF α‐synuclein was significantly associated with survival in the whole prion cohort and the most frequent clinicopathological subtypes. Discussion In the clinical setting, CSF α‐synuclein does not exceed the diagnostic performance of currently used surrogate markers, but it might constitute a robust prognostic indicator.

ObjectiveTo compare the diagnostic accuracy and the prognostic value of blood and cerebrospinal fluid (CSF) tests across prion disease subtypes.MethodsWe used a single-molecule immunoassay to measure tau and neurofilament light chain (NfL) protein levels in the plasma and assessed CSF total(t)-tau, NfL and protein 14-3-3 levels in patients with prion disease (n=336), non-prion rapidly progressive dementias (n=106) and non-neurodegenerative controls (n=37). We then evaluated each plasma and CSF marker for diagnosis and their association with survival, taking into account the disease subtype, which is a strong independent prognostic factor in prion disease.ResultsPlasma tau and NfL concentrations were higher in patients with prion disease than in non-neurodegenerative controls and non-prion rapidly progressive dementias. Plasma tau showed higher diagnostic value than plasma NfL, but a lower accuracy than the CSF proteins t-tau and 14-3-3. In the whole prion cohort, both plasma (tau and NfL) and CSF (t-tau, 14-3-3 and NfL) markers were significantly associated with survival and showed similar prognostic values. However, the intrasubtype analysis revealed that only CSF t-tau in sporadic Creutzfeldt-Jakob disease (sCJD) MM(V)1, plasma tau and CSF t-tau in sCJD VV2, and plasma NfL in slowly progressive prion diseases were significantly associated with survival after accounting for covariates.ConclusionsPlasma markers have lower diagnostic accuracy than CSF biomarkers. Plasma tau and NfL and CSF t-tau are significantly associated with survival in prion disease in a subtype-specific manner and can be used to improve clinical trial stratification and clinical care.

Background In neurodegenerative dementias (NDs) such as prion disease, Alzheimer’s disease (AD), and frontotemporal lobar degeneration (FTLD), protein misfolding leads to the tissue deposition of protein aggregates which, in turn, trigger neuroinflammation and neurodegeneration. Cerebrospinal fluid (CSF) biomarkers have the potential to reflect different aspects of these phenomena across distinct clinicopathological subtypes and disease stages. Methods We investigated CSF glial markers, namely chitotriosidase 1 (CHIT1), chitinase-3-like protein 1 (YKL-40) and glial fibrillary acidic protein (GFAP) in prion disease subtypes (n = 101), AD (n = 40), clinicopathological subgroups of FTLD (n = 72), and controls (n = 40) using validated, commercially available ELISA assays. We explored glial biomarker levels’ associations with disease variables and neurodegenerative CSF biomarkers and evaluated their diagnostic accuracy. The genotype of the CHIT1 rs3831317 polymorphic site was also analyzed. Results Each ND group showed increased levels of CHIT1, YKL-40, and GFAP compared to controls with a difference between prion disease and AD or FTLD limited to YKL-40, which showed higher values in the former group. CHIT1 levels were reduced in both heterozygotes and homozygotes for the CHIT1 24-bp duplication (rs3831317) in FTLD and controls, but this effect was less significant in AD and prion disease. After stratification according to molecular subgroups, we demonstrated (i) an upregulation of all glial markers in Creutzfeldt-Jakob disease VV2 compared to other disease subtypes, (ii) a difference in CHIT1 levels between FTLD with TAU and TDP43 pathology, and (iii) a marked increase of YKL-40 in FTLD with amyotrophic lateral sclerosis (ALS) in comparison with FTLD without ALS. In prion disease, glial markers correlated with disease stage and were already elevated in one pre-symptomatic case of Gerstmann-Sträussler-Scheinker disease. Regarding the diagnostic value, YKL-40 was the only glial marker that showed a moderate accuracy in the distinction between controls and NDs. Conclusions NDs share a CSF profile characterized by increased levels of CSF CHIT1, YKL-40, and GFAP, which likely reflects a common neuroinflammatory response to protein misfolding and aggregation. CSF glial markers of neuroinflammation demonstrate limited diagnostic value but have some potential for monitoring the clinical and, possibly, preclinical phases of NDs.

In this manuscript, we demonstrate the control of film thickness and surface wettability in the photochemical immobilization of poly (vinyl alcohol) (PVA) to a self-assembled monolayer (SAM) containing a phthalimide chromophore. Surface attachment is characterized by ellipsometry and contact angle measurements. The wettability of the resulting films is shown to depend on the chemical composition of the polymer. The film thickness is shown to depend on the irradiation time and molecular weight of the polymer. Using a photomask, micropatterns of polymers can be grafted to the SAM. The photopatterned surface can be "developed" by coating with a thin layer of a mixture containing poly (styrene) (PS) and triphenylsulfonium triflate.

In this study, we performed an ultraviolet-visible (UV-Vis) and circular dichroism (CD) spectroscopic analysis of the binary and ternary supramolecular structures formed by self-assembling the following three water-soluble porphyrins with and without a chiral template: the negatively charged, meso-Tetra(4-sulfonatophenyl) porphine (H2 TPPS4- ); the positively charged meso-trans-(di(N-methyl-4-pyridyl)diphenyl) porphine (trans-DmPyDPP) and meso-cis-(di(N-methyl-4-pyridyl)diphenyl) porphine (cis-DmPyDPP). Polyglutamic acid (both L and D enantiomers) was selected as the chiral template due to its ability to change secondary structure with pH. The propensity for the porphyrins to show an induced CD in the presence of polyglutamic acid is demonstrated. The induced chirality of all supramolecular structures was found to depend on the pH of the solution, the chirality of the polymer, and the order of addition of the positively and negatively charged porphyrins (for ternary complexes). Of particular interest is that the interaction of H2 TPPS4- with the chiral scaffold seems to undergo a dynamic rearrangement of the supramolecular structure as evident from the change in the CD spectrum over time. Moreover, experiments with ternary complexes suggest that the preferential interaction of trans-DmPyDPP with the random coil of the polymer shows promise as a sensor of protein secondary structure.

Prion real-time quaking-induced conversion (RT-QuIC) is emerging as the most potent assay for the in vivo diagnosis of Creutzfeldt–Jakob disease (CJD), but its full application, especially as a screening test, is limited by suboptimal substrate availability, reagent costs, and incomplete assay standardization. Therefore, the search for the most informative cerebrospinal fluid (CSF) surrogate biomarker is still of primary importance. We compared the diagnostic accuracy of CSF protein 14-3-3, measured with both western blot (WB) and enzyme-linked immunosorbent assay (ELISA), total (t)-tau and neurofilament light chain protein (NfL) alone or in combination with RT-QuIC in 212 subjects with rapidly progressive dementia in which we reached a highly probable clinical diagnosis at follow-up or a definite neuropathological diagnosis. T-tau performed best as surrogate CSF biomarker for the diagnosis of CJD (91.3% sensitivity and 78.9% specificity). The 14-3-3 ELISA assay demonstrated a slightly higher diagnostic value compared to the WB analysis (76.9% vs. 72.2%), but both methods performed worse than the t-tau assay. NfL was the most sensitive biomarker for all sCJD subtypes (>95%), including those with low values of t-tau or 14-3-3, but showed the lowest specificity (43.1%). When ELISA-based biomarkers were adopted as screening tests followed by RT-QuIC, t-tau correctly excluded a higher number of non-CJD cases compared to NfL and 14-3-3 ELISA. Our study showed that among the CSF surrogate biomarkers of potential application for the clinical diagnosis of CJD, t-tau performs best either alone or as screening test followed by RT-QuIC as a second-level confirmatory test.

he article Ultrasensitive RT‑QuIC assay with high sensitivity and specificity for Lewy body‑

In this manuscript we present a spectroscopic study of the supramolecular interaction of photo-switchable 4,4′-Azobenzene dicarboxylic acid (ADA) with L and D poly-Glutamic acid (Poly-Glu). ADA is water-soluble and undergoes photo-isomerisation between trans and cis geometries when irradiated with UV and visible light. Non-covalent assembly of the photo-switch with the polypeptide is induced by reducing the pH of an aqueous solution containing both the ADA and Poly-Glu and characterized with circular dichroism (CD) and UV–vis spectroscopy. The properties of the aggregates depend on multiple factors such as the molecular conformation of the photo-switch, which is selected with UV and visible light, the pH of the solution and the manner in which the pH is reduced (rapid vs. slow reduction). The polypeptide acts as a chiral template for trans-ADA chromophores as demonstrated by the appearance of an induced CD signal of the achiral photo-switch in the presence of Poly-L-Glu at reduced pH. In neutral aqueous solutions trans-ADA can be photo-isomerised to cis-ADA, however, its aggregated form loses this capability. In contrast to the trans geometry, CD and UV–vis spectroscopy show that Poly-Glu is unable to function as a chiral template during the aggregation of cis-ADA. Nevertheless, the polymer is able to induce chirality when cis-ADA aggregates are photo-isomerised to the trans form by irradiation with visible light, as shown by the appearance of an induced CD signal. Distinct differences observed in the CD and UV–vis spectra depending on the mode of pH reduction, indicates that assembly occurs under hierarchical control.

In this report we present a UV–Vis spectroscopic study of the pH dependent optical properties of 4,4′-azobenzene dicarboxylic acid in aqueous solution. A combination of chemical (acid-base) and light stimuli is combined to demonstrate that the system undergoes two types of optical switching cycles. At neutral pH the azobenzene undergoes photo-induced cis-trans isomerisation. Upon pH reduction the UV–Vis spectra show changes consistent with aggregation of the azobenzene photo-switch. The photo-responsive behaviour is dependent on the pH and conformation of the azobenzene. The optical properties of the system are dependent on the mode of pH reduction and the isomeric cis/trans composition of the photo-switch, showing hierarchical control of self-assembly.

J aggregates of the tetraanionic meso-tetrakis(4-sulfonatophenyl)porphine (H2TPPS4) gain chirality by interaction with ?- or ?-[Ru] ([Ru]=[Ru(1,10-phenanthroline)3]2+). After destroying and reforming the aggregates by changing the pH of the solution, the final species "remember" the initially imprinted chirality, even in the presence of an excess of the ruthenium complex with the opposite chirality to that memorized.

We describe the synthesis and characterization of a series of water-soluble free-base, zinc, and copper porphyrin-oligonucleotide (ODN) conjugates. A non-charged tetraarylporphyrin was directly attached to the 5'-position of thymine via a short amide linker. Such a linker should allow for rigid connection to the adjacent nucleobases, thus increasing the sensitivity for monitoring conformational changes of the ODNs by electronic circular dichroism due to capping effects or ligand binding. Two complementary synthetic approaches have been used to incorporate porphyrin chromophores into the DNA. In the first approach a porphyrin carboxylic acid is conjugated to 5'-amino-ODN. In the second approach the phosphoramidite of the porphyrin-amido-thymidine is coupled to 5'-hydroxy-ODN using standard automated phosphoramidite chemistry. In both cases a spontaneous metallation of the free-base porphyrin in porphyrin-DNA conjugates was observed during the porphyrin-DNA conjugate cleavage from the solid support and its consequent deprotection using concentrated aqueous ammonia. Zinc and copper porphyrin-DNA conjugates were isolated by HPLC and characterized together with the anticipated free-base porphyrin-DNA conjugate. Authentic zinc and copper porphyrin-DNA conjugates were intentionally prepared from intentionally metallated porphyrins to compare their spectroscopic and HPLC characteristics with isolated metallospecies and to confirm the spontaneous metallation.

Molecular systems containing two or more interacting porphyrins show remarkable spectroscopic features that allow for a very sensitive detection of conformational changes on the microscale level by different methods, such as fluorescence and electronic circular dichroism (ECD). Covalent porphyrin-DNA assemblies can provide a CD profile (exciton couplet) within the porphyrin Soret band region which is very diagnostic for DNA conformational changes. Additionally, covalently linked porphyrins have been shown to function as DNA molecular caps and to stabilize the non-self-complementary non-Watson-Crick guanine-adenine DNA sequence via their strong π-π stacking.

This report demonstrates the electroless deposition of Ni onto micropatterns of poly (acrylic acid) (PAA) photografted to phthalimide-terminated self-assembled monolayers (SAMs). PAA is spin-coated onto phthalimide SAMs and covered with a photomask. UV irradiation selectively binds PAA to exposed regions of the surface, allowing PAA on unexposed regions to be rinsed off. A Pd catalyst is then selectively adsorbed to regions of the surface where PAA is bound. The adsorbed catalyst selectively initiates Ni plating upon immersion of the substrate into a Ni(SO4) bath.

The interesting structural, electronic, and optical properties of DNA provide fascinating opportunities for developing nanoscale smart materials by integrating DNA with opto-electronic components. In this article we demonstrate the electrostatic binding of an amine-terminated dithienylethene (DET) molecular switch to double-stranded synthetic polynucleotides. The DET switch can undergo photochemical ring-closure and opening reactions. Circular dichroism (CD) and UV-vis spectroscopy show that both the open, 1o, and the closed, 1c, forms of the switch bind to DNA. Upon addition of DNA to a solution of 1o or 1c, the UV-vis spectrum displays a hypochromic effect, indicative of an interaction between the switch and the DNA. The chirality of the DNA double helix is transmitted to the switching unit which displays a well-defined CD signal upon supramolecular complexation to the DNA. Additionally, the CD signal of the DNA attenuates, demonstrating that both components of the complex mutually influence each other's structure; the DNA induces chirality in the switch, and the switch modifies the structure of the DNA. Modulation of the chiroptical properties of the complex is achieved by photochemically switching the DET between its ring open and closed isomers. A pH dependence study of the binding shows that when the pH is increased the switches lose their binding ability, indicating that electrostatic interactions between protonated amines and the negatively charged phosphate backbone are the dominant driving force for binding to the DNA. A comparison of poly(deoxyguanylic-deoxycytidylic) acid [poly(dGdC)(2)] polynucleotides with poly(deoxyadenylic-deoxythymidylic) acid [poly(dAdT)(2)] shows distinct differences in the CD spectra of the complexes.

Transfer of chirality is an intriguing issue worth studying to understand better the origin of life and for possible technological applications. In the last few years we have been working in this area studying the chain of events that begins with induction, reaches a permanent transfer (chiral memory) and extends in some cases to a (quasi-)reversible situation in which induced and permanently memorized chirality coexists. This can happen thanks to a designed blend of thermodynamics and kinetics.

We have explored the utility, strength, and limitation of through- space exciton-coupled circular dichro- ism in determination of the secondary structure of optically active chromo- phoric nanoarrays using the example of end-capped porphyrin- and metallo- porphyrin-oligodeoxynucleotide conju- gates. We put special emphasis on the explanation of the origin and signifi- cance of the distinctive multiple bands in the CD spectra (trisignate and tetra- signate CD bands). Such CD profiles are often observed in chiral aggregates or multichromophoric arrays but have never before been studied in detail. We found that variation of temperature and ionic strength has a profound effect on the geometry of the porphy- rin-DNA conjugates and thus the nature of electronic interactions. At lower temperatures and in the absence of NaCl all three 5'-DNA-porphyrin conjugates display negative bisignate CD exciton couplets of variable inten- sity in the Soret region resulting from through-space interaction between the electric transition dipole moments of the two end-capped porphyrins. As the temperature is raised these exciton couplets are transformed into single positive bands originating from the porphyrin-single-strand DNA interac- tions. At higher ionic strengths and low temperatures, multisignate CD bands are observed in the porphyrin Soret region. These CD signature bands orig- inate from a combination of intermo- lecular, end-to-end porphyrin-porphy- rin stacking between duplexes and por- phyrin-DNA interactions. The inter- molecular aggregation was confirmed by fluorescence and absorption spec- troscopy and resonance light scattering. DeVoe theoretical CD calculations, in conjunction with molecular dynamics simulations and Monte Carlo confor- mational searches, were used to mimic the observed bisignate exciton-coupled CD spectra as well as multiple CD bands. Calculations correctly predicted the sign and shape of the experimental- ly observed CD spectra. These studies reveal that the exciton-coupled circular dichroism is a very useful technique for the determination of the structure of optically active arrays.

The anionic nickel(II) porphyrin NiTPPS is able to selectively sense the spermine induced left-handed Z-form of DNA while it is completely silent in the presence of right-handed B-DNA. Interactions between the DNA and the porphyrin can be easily modulated by pH and temperature. The resulting Z-DNA−porphyrin−spermine complex behaves as a supramolecular reversible information storage system and as a reversible AND logic gate.