Synthesis, Characterization and Surface Group Quantification of Functionalized Polymer Particles for Signal Amplification Strategies

Polymer nanoparticles (PNP) are increasingly used as tools in (bio)analytics. Typical applications of PNP include carriers for drugs, carriers for dye molecules for signal amplification in optical assays, nanosensors and targeted probes for bioimaging studies. These applications in life sciences impose stringent requirements on particle size, size distribution, morphology, colloidal stability, biocompatibility, optical properties, and ease of surface functionalization with, for example, targeting ligands, sensor molecules, and linkers. PNP, including the polystyrene particles (PSP) which are in the focus of this work, are non-fluorescent by nature but can be made fluorescent with the aid of luminophores such as organic dyes. Fluorescent PNP can be obtained by coupling reactive fluorophores to the surface groups of the PNP or by encapsulation of fluorophores into the PNP matrix. The latter approach is particularly attractive due to its versatility since the encapsulation into preformed PNP does not require luminophores with reactive functional groups (FGs), but rather only requires hydrophobic luminophores. Moreover, the fluorophores are protected from the potentially fluorescence quenching environment surrounding the PNP matrix and the reactive groups on the PNP surface can be exclusively used for the attachment of targeting ligands. Dye loading of PNP typically requires a dye-specific optimization of the loading concentration with respect to signal strength as conventional dyes commonly form barely emissive or even non-fluorescent aggregates at high loading concentrations. Exceptions exist, which are dyes with propeller-like groups that show aggregation-induced emission (AIE). These dyes can be loaded with high concentrations into PNP without detrimental fluorescence quenching effects and can even exhibit fluorescence and hence signal amplification upon aggregation. Due to these attractive properties, AIE dyes for use in PNP were investigated. The spectroscopic properties of different AIE dye derivatives were systematically studied in organic solvents, solvent−water mixtures, and in the solid state. Dyes with optimal performance were entrapped in PSP. The staining of PSP with these AIE dyes resulted in a considerable increase in the dye fluorescence quantum yield and lifetime, reflecting the combined influence of the restricted molecular motion and the reduced polarity of the dye microenvironment. Functionalization of undoped and dye loaded PNP with, for example, targeting ligands requires knowledge of the chemical nature and total amount of the surface groups as well as the amount of surface FGs accessible for coupling reactions such as the conjugation of biomolecules. These numbers can differ considerably depending on factors including particle morphology and sterical constraints. Ideal methods for surface group quantification should be robust, reliable, and fast. Moreover, they should not require expensive instrumentation and should be versatile to enable the characterization of a broad variety of particle systems independent of their optical properties, including systems that scatter or include systems with encoded dyes. For this respect, we studied a variety of optical assays for the quantification of carboxy and amino surface groups commercial and custom-made particles with varying surface group densities. We performed these studies using both conventional reporters such as fluorescein derivatives, Fluram, or IR 797 as well as synthetically customized cleavable labels. Special emphasis was dedicated to the development of a platform of cleavable and multimodal labels which consist of a suitable reactive group such as NHS-esters or amine, a quantitatively cleavable linker such as disulfide, and an optically active moiety such as 2-thiopyridone for optical assays. Conventional reporters are measured when bound to the particle surface, which renders the resulting optical signals prone to distortions by scattering and interferences from encoding dyes. In contrast, these cleavable labels can be detected photometrically or fluorometrically in the supernatant after quantitative cleavage of the linker. Moreover, the linker unit is designed in such a way that the products of the cleaved linkers remaining at the particle surface can also be detected optically. In addition, the presence of a heteroatom such as sulfur, nitrogen or fluorine in the reporter and/or the linker can be detected by an analytical method relying on a different measurement principle. This allows for straightforward validation by method comparison with, for instance, ICP-OES. Thereby, FGs on a broad variety of different particles such as PNP, silica nanoparticles (NPs), and metal particles can also be quantified

Nitrous oxide-induced motor-predominant axonal peripheral neuropathy: A phenotype distinct from isolated vitamin B12 deficiency

Highlights: N2O toxicity and B12 deficiency both cause a sensory predominant myeloneuropathy. N2O abuse may also lead to a selectively severe motor axonal peripheral neuropathy. This motor neuropathy phenotype may be related to N2O direct neural toxicity. In-vivo measures of motor axonal dysfunction are more pronounced in N2O abuse

First presentation of LPIN1 acute rhabdomyolysis in adolescence and adulthood

LPIN1 mutations are a known common cause of autosomal recessive, recurrent and life-threatening acute rhabdomyolysis of childhood-onset. The first episode of rhabdomyolysis usually happens in nearly all cases before the age of 5 and death is observed in 1/3 of patients. Here we present two cases of acute rhabdomyolysis with a milder phenotype caused by LPIN1 mutation presenting in adolescence (11 years old) and adulthood (40 years old) after Parvovirus infection and metabolic stress, respectively. In our opinion, the mutation types, epigenetic factors, the environment exposition to triggers or the existence of proteins with a similar structure of LPIN1, may have a role in modulating the onset of rhabdomyolysis. LPIN1 should be included on a panel of genes analysed in the investigation of adult individuals with rhabdomyolysis. Metabolic and viral stressors should be included in the list of possible rhabdomyolysis precipitant

Mitochondrial Neurogastrointestinal Encephalomyopathy: Into the Fourth Decade, What We Have Learned So Far.

Mitochondrial neurogastrointestinal encephalomyopathy (MNGIE) is an ultra-rare metabolic autosomal recessive disease, caused by mutations in the nuclear gene TYMP which encodes the enzyme thymidine phosphorylase. The resulting enzyme deficiency leads to a systemic accumulation of the deoxyribonucleosides thymidine and deoxyuridine, and ultimately mitochondrial failure due to a progressive acquisition of secondary mitochondrial DNA (mtDNA) mutations and mtDNA depletion. Clinically, MNGIE is characterized by gastrointestinal and neurological manifestations, including cachexia, gastrointestinal dysmotility, peripheral neuropathy, leukoencephalopathy, ophthalmoplegia and ptosis. The disease is progressively degenerative and leads to death at an average age of 37.6 years. As with the vast majority of rare diseases, patients with MNGIE face a number of unmet needs related to diagnostic delays, a lack of approved therapies, and non-specific clinical management. We provide here a comprehensive collation of the available knowledge of MNGIE since the disease was first described 42 years ago. This review includes symptomatology, diagnostic procedures and hurdles, in vitro and in vivo disease models that have enhanced our understanding of the disease pathology, and finally experimental therapeutic approaches under development. The ultimate aim of this review is to increase clinical awareness of MNGIE, thereby reducing diagnostic delay and improving patient access to putative treatments under investigation

Safety and Efficacy of Erythrocyte Encapsulated Thymidine Phosphorylase in Mitochondrial Neurogastrointestinal Encephalomyopathy.

Mitochondrial neurogastrointestinal encephalomyopathy (MNGIE) is an ultra-rare autosomal recessive disorder of nucleoside metabolism that is caused by mutations in the nuclear thymidine phosphorylase gene (TYMP) gene, encoding for the enzyme thymidine phosphorylase. There are currently no approved treatments for MNGIE. The aim of this study was to investigate the safety, tolerability, and efficacy of an enzyme replacement therapy for the treatment of MNGIE. In this single centre study, three adult patients with MNGIE received intravenous escalating doses of erythrocyte encapsulated thymidine phosphorylase (EE-TP; dose range: 4 to 108 U/kg/4 weeks). EE-TP was well tolerated and reductions in the disease-associated plasma metabolites, thymidine, and deoxyuridine were observed in all three patients. Clinical improvements, including weight gain and improved disease scores, were observed in two patients, suggesting that EE-TP is able to reverse some aspects of the disease pathology. Transient, non-serious adverse events were observed in two of the three patients; these did not lead to therapy discontinuation and they were managed with pre-medication prior to infusion of EE-TP. To conclude, enzyme replacement therapy with EE-TP demonstrated biochemical and clinical therapeutic efficacy with an acceptable clinical safety profile

Prevalence and incidence of neuromuscular conditions in the UK between 2000 and 2019: A retrospective study using primary care data.

BACKGROUND: In the UK, large-scale electronic primary care datasets can provide up-to-date, accurate epidemiological information on rarer diseases, where specialist diagnoses from hospital discharges and clinic letters are generally well recorded and electronically searchable. Current estimates of the number of people living with neuromuscular disease (NMD) have largely been based on secondary care data sources and lacked direct denominators. OBJECTIVE: To estimate trends in the recording of neuromuscular disease in UK primary care between 2000-2019. METHODS: The Clinical Practice Research Datalink (CPRD) database was searched electronically to estimate incidence and prevalence rates (per 100,000) for a range of NMDs in each year. To compare trends over time, rates were age standardised to the most recent CPRD population (2019). RESULTS: Approximately 13 million patients were actively registered in each year. By 2019, 28,230 active patients had ever received a NMD diagnosis (223.6), which was higher among males (239.0) than females (208.3). The most common classifications were Guillain-Barre syndrome (40.1), myasthenia gravis (33.7), muscular dystrophy (29.5), Charcot-Marie-Tooth (29.5) and inflammatory myopathies (25.0). Since 2000, overall prevalence grew by 63%, with the largest increases seen at older ages (≥65-years). However, overall incidence remained constant, though myasthenia gravis incidence has risen steadily since 2008, while new cases of muscular dystrophy fell over the same period. CONCLUSIONS: Lifetime recording of many NMDs on primary care records exceed current estimates of people living with these conditions; these are important data for health service and care planning. Temporal trends suggest this number is steadily increasing, and while this may partially be due to better recording, it cannot be simply explained by new cases, as incidence remained constant. The increase in prevalence among older ages suggests increases in life expectancy among those living with NMDs may have occurred

Different attachment styles correlate with mood disorders in adults with epilepsy or migraine.

PURPOSE: Interpersonal relationships are viewed as important contexts within which psychopathology emerges and persists or desists. Attachment theory describes the dynamics of long-term relationships between humans especially in families and lifelong friendships. The present study was aimed at investigating attachment styles in adult patients with epilepsy as compared to subjects with migraine and their potential correlates with a history of mood disorders. METHODS: A consecutive sample of 219 adult outpatients with epilepsy (117) or migraine (102) was assessed with the Attachment Style Questionnaire (ASQ). RESULTS: Patients with epilepsy and a lifetime history of mood disorders presented elevated scores for Need for approval (p<0.001) and Preoccupation with relationships (p<0.001). Age correlated with the Relationships as secondary (r=0.322; p<0.001) and Need for approval (r=0.217; p=0.019) subscales while age at onset correlated only with Relationships as secondary (r=0.225; p=0.015). Seizure-free patients presented lower scores for Need for approval (p=0.003). Patients with migraine and a lifetime history of mood disorders presented lower scores in Confidence (p=0.002) and higher scores in Discomfort with closeness (p=0.026). CONCLUSIONS: An anxious-preoccupied attachment correlated with mood disorders in epilepsy while it was an avoidant pattern in migraine. Our results bring further data on the role of psychological variables in mood disorders in epilepsy. Further studies will allow early identification of patients at risk and the development of preventive strategies