Identification of protein-based materials in Cultural Heritage: immunodetection of paint binders

This PhD thesis aims to investigate the identification of proteinaceous materials in particular used in paintings and artworks by immunodetection and other complimentary methodologies. The thesis is divided into two parts. The first part (chapters 1-3) goes in depth on the detection of proteins with ELISA and its optimization with silica nanoparticles. The second (chapters 4- 5) investigates the development of paint model replicas to imitate real artworks with proteinaceous binders, application of other complimentary techniques to detect proteins and the comparison with ELISA on real artworks. In particular, chapter 1 focuses the use of proteins in artworks and bibliographical literature on the particular use of ELISA as a method for detection and investigates the different complimentary techniques such as stratigraphy, Naphthalene Blue black and Pyrolysis GC-MS and how they have been applied in the past to detect proteins in artworks and the use of standard markers for characterization with chromatography. Chapter 2 provides an in-depth case study of ELISA based immunoassays to detect proteins from previously developed paint models. Chapter 3 investigates the optimisation of ELISA with silica nanoparticles for protein detection. Chapter 4 illustrates the development of paint model replicas with the use of heritage materials and proteinaceous binders and their characterization to detect proteins with Pyrolysis GC-MS. Chapter 5 portrays four case studies from the samples obtained at the Perpignan museum in France and their characterization with stratigraphy, Pyrolysis GC-MS and ELISA while comparing the results of protein detection between the latter two; Resumo: Identificação de materiais à base de proteínas no Património Cultural: Imunodetecção de ligantes de tintas Este projeto visa investigar a identificação de materiais proteicos, nomeadamente utilizados em pinturas e obras de arte pora imunodetecção e outras metodologias complementares. A tese está dividida em duas partes. A primeira parte (capítulos 1-3) desenvolve de forma detalhada a deteção de proteínas com ELISA e a sua otimização com nanopartículas de sílica. A segunda parte (capítulos 4-6) investiga o desenvolvimento de réplicas de modelos de pintura que mimetizam obras reais, com diferentes ligandos proteicos, e utilizam-se diversas técnicas complementares para detetar proteínas. Faz-se ainda uma comparação com a deteção por ELISA em obras de arte reais. Em particular, o capítulo 1 centra-se na utilização de proteínas em obras de arte e literatura bibliográfica sobre a utilização particular da ELISA como método de deteção. O capítulo 2 fornece um estudo aprofundado de caso de imunoensaios baseados em ELISA para detetar proteínas de modelos de pintura previamente desenvolvidos. O Capítulo 3 investiga a otimização da ELISA com nanopartículas de sílica para deteção de proteínas. O capítulo 4 investiga as diferentes técnicas complementares, tais como a estratigrafia, a naftalina azul e a pirólise GC-MS e como foram aplicadas no passado para detetar proteínas em obras de arte e a utilização de marcadores padrão para caracterização com cromatografia. O Capítulo 5 ilustra o desenvolvimento de réplicas de modelos de pintura com a utilização de materiais patrimoniais e aglutinantes proteicos e a sua caracterização para detetar proteínas com Pirólise GC-MS. O capítulo 6 retrata quatro estudos de caso das amostras obtidas no museu de Perpignan em França e a caracterização com estratigrafia, Pirólise GCMS e ELISA, comparando os resultados da deteção de proteínas entre os dois últimos

Binocular vision parameters in chronic heavy alcoholics: Short-term outcomes of alcohol detoxification

Background: Alcohol consumption is rising in developing countries such as India, and alcohol addiction has systemic and ocular impacts. This study aimed to investigate the binocular functions of chronic heavy alcoholics before and after alcohol detoxification. Methods: A prospective before–after study was designed and conducted at Treda De-Addiction Centre, Bengaluru, India. Males in the age range of 30–40 years who had been alcohol addicts for more than six years and met the inclusion criteria were recruited. We performed a routine optometric examination followed by detailed binocular vision assessment, including accommodative, vergence, and oculomotor tests on the first day of rehabilitation and one month after initiation of rehabilitation. Results: Twenty-five males with of the age (mean ± standard deviation [SD]) 36.24 ± 4.33 years were evaluated. The pre- and post-detoxification mean ± SD of the monocular (right eye: 5.98 ± 3.50, 6.60 ± 3.49; left eye: 6.18 ± 3.69, 7.10 ± 3.78) and binocular accommodative facility (7.10 ± 3.93, 7.40 ± 4.51) did not change significantly (all P > 0.05). Eighteen (72%) of the participants had non-strabismic binocular vision anomalies (NSBVA), among whom the accommodative infacility and convergence insufficiency were higher in frequency and remained constant after alcohol detoxification. Furthermore, the binocular vision parameters showed no statistically significant difference between the pre- and post-detoxification values (all P > 0.05). Conclusions: The binocular vision parameters did not change significantly after one month of alcohol detoxification in the chronic heavy drinkers. Most long-term alcoholics had NSBVA with no change after rehabilitation, indicating that short-term alcohol detoxification may not aid in the recovery of binocular parameters. However, further validation is required. Assessing the impact of vision therapy in addition to a longer period of abstinence can confirm or refute the persistence of observed effects of chronic alcohol consumption on binocular functions and NSBVA in this population

Probing lens-induced gravitational-wave birefringence as a test of general relativity

Theories beyond general relativity (GR) modify the propagation of gravitational waves (GWs). In some, inhomogeneities (aka. gravitational lenses) allow interactions between the metric and additional fields to cause lens-induced birefringence (LIB): a different speed of the two linear GW polarisations ($+$ and $\times$). Inhomogeneities then act as non-isotropic crystals, splitting the GW signal into two components whose relative time delay depends on the theory and lens parameters. Here we study the observational prospects for GW scrambling, i.e when the time delay between both GW polarisations is smaller than the signal's duration and the waveform recorded by a detector is distorted. We analyze the latest LIGO-Virgo-KAGRA catalog, GWTC-3, and find no conclusive evidence for LIB. The highest log Bayes factor that we find in favour of LIB is $3.21$ for GW$190521$, a particularly loud but short event. However, when accounting for false alarms due to (Gaussian) noise fluctuations, this evidence is below 1-$\sigma$. The tightest constraint on the time delay is $<0.51$ ms (90% C.L.) from GW$200311\_115853$. From the non-observation of GW scrambling, we constrain the optical depth for LIB, accounting for the chance of randomly distributed lenses (eg. galaxies) along the line of sight. Our LIB constraints on a (quartic) scalar-tensor Horndeski theory are more stringent than solar system tests for a wide parameter range and comparable to GW170817 in some limits. Interpreting GW190521 as an AGN binary (i.e. taking an AGN flare as a counterpart) allows even more stringent constraints. Our results demonstrate the potential and high sensitivity achievable by tests of GR, based on GW lensing.Comment: 18 pages, 10 figure

Linear cyclodextrin polymer prodrugs as novel yherapeutics for Niemann-Pick type C1 disorder

Niemann-Pick Type C1 disorder (NPC) is a rare lysosomal storage disease characterized by the accumulation of cholesterol in lysosomes. NPC has no FDA approved treatments yet, however 2-hydroxypropyl-β-cyclodextrin (HPβCD) has shown efficacy for treating the disease in both mouse and feline NPC models and is currently being investigated in late stage clinical trials. Despite promising results, therapeutic use of HPβCD is limited by the need for high doses, ototoxicity and intrathecal administration. These limitations can be attributed to its poor pharmacokinetic profile. In the attempt to overcome these limitations, we have designed a β-cyclodextrin (βCD) based polymer prodrugs (ORX-301) for an enhanced pharmacokinetic and biodistribution profile, which in turn can potentially provide an improved efficacy at lower doses. We demonstrated that subcutaneously injected ORX-301 extended the mean lifespan of NPC mice at a dosage 5-fold lower (800 mg/kg, body weight) the HPβCD dose proven efficacious (4000 mg/kg). We also show that ORX-301 penetrates the blood brain barrier and counteracts neurological impairment. These properties represent a substantial improvement and appear to overcome major limitations of presently available βCD-based therapy, demonstrating that this novel prodrug is a valuable alternative/complement for existing therapies

Mesoporous Silica Based Protein Release Systems

Mesoporous silica nanoparticles (MSNs) such as MCM41 are effective support carriers with excellent adsorption properties and large surface areas [1-3]. Bioactive molecules like proteins such as albumin, casein and collagen are universally present in nature and products. Interaction of these proteins and MCM41 have not been widely explored. The aim of this study is to assess how these proteins behave in the presence of MCM41 and assess its protein release properties for the purpose of interdisciplinary applications

Optimization of protein extraction and ELISA immunodetection from protein-based paint models with mesoporous silica nanoparticles and MCM41

Protein-based biological materials such as albumin, casein and collagen are found in various cultural heritage (CH) artefacts. This study focuses on the study of protein binders from easel paintings media. Proteins have complex structures which are difficult to identify with non-invasive spectroscopic methods (FT-IR, Raman, UV). Immunoassays such as ELISA determine the protein’s source of origin which is necessary for art objects. To increase the detection and identification of proteins by immunoassays, the efficiency of micro-extraction of proteins from heritage materials is a crucial step. Extractions mediated by cycles of orbital agitation and ultrasonic radiation give the possibility to extract proteins from easel painting sample. In this work, protein-based paint models coupled with silica nanoparticles were used for micro-extraction. Nanoparticles possess high surface-to-volume ratios that can attach bioactive molecules such as proteins and increase the total protein recovered from microsamples. Protein extracts were quantified with Bradford Assay in the presence of Coomassie blue. The protein recovery results were statistically computed, and the SPSS analysis shows significant (p <0.05) increase in protein recovery, above 1.3 times for NPSiO2 and above 1.6 times for MCM-41. The statistical data shows evidence that silica nanoparticles intensify the total protein recovered from paint microsamples. Finally, ELISA was realized on the protein extracts to verify and compare the immunodetection of protein from the paint models with and without the use of silica nanoparticles

Post-intervention Status in Patients With Refractory Myasthenia Gravis Treated With Eculizumab During REGAIN and Its Open-Label Extension

OBJECTIVE: To evaluate whether eculizumab helps patients with anti-acetylcholine receptor-positive (AChR+) refractory generalized myasthenia gravis (gMG) achieve the Myasthenia Gravis Foundation of America (MGFA) post-intervention status of minimal manifestations (MM), we assessed patients' status throughout REGAIN (Safety and Efficacy of Eculizumab in AChR+ Refractory Generalized Myasthenia Gravis) and its open-label extension. METHODS: Patients who completed the REGAIN randomized controlled trial and continued into the open-label extension were included in this tertiary endpoint analysis. Patients were assessed for the MGFA post-intervention status of improved, unchanged, worse, MM, and pharmacologic remission at defined time points during REGAIN and through week 130 of the open-label study. RESULTS: A total of 117 patients completed REGAIN and continued into the open-label study (eculizumab/eculizumab: 56; placebo/eculizumab: 61). At week 26 of REGAIN, more eculizumab-treated patients than placebo-treated patients achieved a status of improved (60.7% vs 41.7%) or MM (25.0% vs 13.3%; common OR: 2.3; 95% CI: 1.1-4.5). After 130 weeks of eculizumab treatment, 88.0% of patients achieved improved status and 57.3% of patients achieved MM status. The safety profile of eculizumab was consistent with its known profile and no new safety signals were detected. CONCLUSION: Eculizumab led to rapid and sustained achievement of MM in patients with AChR+ refractory gMG. These findings support the use of eculizumab in this previously difficult-to-treat patient population. CLINICALTRIALSGOV IDENTIFIER: REGAIN, NCT01997229; REGAIN open-label extension, NCT02301624. CLASSIFICATION OF EVIDENCE: This study provides Class II evidence that, after 26 weeks of eculizumab treatment, 25.0% of adults with AChR+ refractory gMG achieved MM, compared with 13.3% who received placebo

Minimal Symptom Expression' in Patients With Acetylcholine Receptor Antibody-Positive Refractory Generalized Myasthenia Gravis Treated With Eculizumab

The efficacy and tolerability of eculizumab were assessed in REGAIN, a 26-week, phase 3, randomized, double-blind, placebo-controlled study in anti-acetylcholine receptor antibody-positive (AChR+) refractory generalized myasthenia gravis (gMG), and its open-label extension