Advances in Nucleotide Repeat Expansion Diseases: Transcription Gets in Phase.

Unstable DNA repeat expansions and insertions have been found to cause more than 50 neurodevelopmental, neurodegenerative, and neuromuscular disorders. One of the main hallmarks of repeat expansion diseases is the formation of abnormal RNA or protein aggregates in the neuronal cells of affected individuals. Recent evidence indicates that alterations of the dynamic or material properties of biomolecular condensates assembled by liquid/liquid phase separation are critical for the formation of these aggregates. This is a thermodynamically-driven and reversible local phenomenon that condenses macromolecules into liquid-like compartments responsible for compartmentalizing molecules required for vital cellular processes. Disease-associated repeat expansions modulate the phase separation properties of RNAs and proteins, interfering with the composition and/or the material properties of biomolecular condensates and resulting in the formation of abnormal aggregates. Since several repeat expansions have arisen in genes encoding crucial players in transcription, this raises the hypothesis that wide gene expression dysregulation is common to multiple repeat expansion diseases. This review will cover the impact of these mutations in the formation of aberrant aggregates and how they modify gene transcription.

Pentanucleotide repeat-related disorders: Genetics and bioinformatic discovery and detection.

In recent years, a large group of familial epilepsies and hereditary ataxias have emerged, caused by an extraordinary type of a novel pentanucleotide repeat expansion that has arisen in a preexisting nonpathogenic repeat tract. Remarkably, these insertions have occurred in noncoding regions of genes expressed in the cerebellum, but with highly diverse functions. These conditions, clinically very heterogeneous, may remain underdiagnosed in patients with atypical phenotypes and age at onset. They share, however, many genetic and phenotypic features, and discovery or detection of their pathogenic pentanucleotide repeats for diagnostic purposes can be achieved using recent bioinformatic methods. Here, we focus on the latest advances regarding the peculiar group of pentanucleotide repeat-related disorders beyond epilepsies.

Familial Cerebellar Ataxia and Amyotrophic Lateral Sclerosis/Frontotemporal Dementia with DAB1 and C9ORF72 Repeat Expansions: An 18-Year Study.

BACKGROUND: Coding and noncoding repeat expansions are an important cause of neurodegenerative diseases. OBJECTIVE: This study determined the clinical and genetic features of a large German family that has been followed for almost 2 decades with an autosomal dominantly inherited spinocerebellar ataxia (SCA) and independent co-occurrence of amyotrophic lateral sclerosis (ALS) and frontotemporal dementia (FTD). METHODS: We carried out clinical examinations and telephone interviews, reviewed medical records, and performed magnetic resonance imaging and positron emission tomography scans of all available family members. Comprehensive genetic investigations included linkage analysis, short-read genome sequencing, long-read sequencing, repeat-primed polymerase chain reaction, and Southern blotting. RESULTS: The family comprises 118 members across seven generations, 30 of whom were definitely and five possibly affected. In this family, two different pathogenic mutations were found, a heterozygous repeat expansion in C9ORF72 in four patients with ALS/FTD and a heterozygous repeat expansion in DAB1 in at least nine patients with SCA, leading to a diagnosis of DAB1-related ataxia (ATX-DAB1; SCA37). One patient was affected by ALS and SCA and carried both repeat expansions. The repeat in DAB1 had the same configuration but was larger than those previously described ([ATTTT]≈75 [ATTTC]≈40-100 [ATTTT]≈415 ). Clinical features in patients with SCA included spinocerebellar symptoms, sometimes accompanied by additional ophthalmoplegia, vertical nystagmus, tremor, sensory deficits, and dystonia. After several decades, some of these patients suffered from cognitive decline and one from additional nonprogressive lower motor neuron affection. CONCLUSION: We demonstrate genetic and clinical findings during an 18-year period in a unique family carrying two different pathogenic repeat expansions, providing novel insights into their genotypic and phenotypic spectrums. © 2022 The Authors. Movement Disorders published by Wiley Periodicals LLC on behalf of International Parkinson and Movement Disorder Society.

Lipoplexes and polyplexes as nucleic acids delivery nanosystems: The current state and future considerations.

INTRODUCTION: Designing safe and effective nucleic acid delivery nanosystems presents a challenge that requires a good understanding of various biological barriers, whose impact is frequently neglected during in vitro assessments. Hence, the development of nanosizing non-viral vectors would benefit from a more thorough physicochemical characterization to establish structure-activity relationships and increase the preclinical data relevance. AREAS COVERED: This review focused on major barriers of lipoplexes and polyplexes by systemic delivery such as blood and immune cells and is aimed to serve as a prescreening tool for the fast and safe development of both non-viral vectors in vivo. An outline of the preclinical assays to be performed under physiological representative conditions, to better account for or even predict the highly dynamic interactions in humans is also given. EXPERT OPINION: The rational design of non-viral vectors has shown promising intracellular uptake results in vitro. Translating in vitro success into clinics has gone with progress, but it is still a difficult task to achieve, and more closely mimicking biological environment in vitro assays of lipoplexes and polyplexes may provide more correlated results to in vivo experiments. Clinical practice would benefit from safer non-viral vectors, particularly when avoiding patients' immune responses and toxicity, which is of major concern.

La formación del operario en vigilancia y lucha antivectorial. Matanzas, 2019

RESUMEN Introducción: para la formación de operarios integrales en vigilancia y lucha antivectorial, está diseñado un curso de habilitación. El claustro de profesores de la provincia de Matanzas ha encontrado algunas dificultades en la adquisición de conocimientos por parte de los estudiantes. Objetivo: caracterizar la fuerza laboral de vigilancia y lucha antivectorial de la provincia de Matanzas, para diseñar un conjunto de indicaciones que complementen los contenidos del curso de habilitación de operarios, de forma que los estudiantes se sientan motivados y entiendan mejor los contenidos ya incluidos. Materiales y métodos: se realizó una investigación descriptiva, retrospectiva y longitudinal, de enero a diciembre de 2019. Resultados: el grupo de trabajadores de los operarios es el más numeroso. El nivel educacional que predominó fue el de secundaria básica. En 2019 se realizaron 17 cursos de formación, en cuyas evaluaciones el claustro de profesores encontró dificultades en las respuestas a las preguntas de tres de los temas del curso, al igual que en el uso de la lengua materna. Conclusiones: se propuso introducir contenidos en cada tema del curso de formación, que se impartirán de acuerdo a las formas de enseñanza y aprendizaje ya definidas en el programa. El profesor responsable del tema lo dará en el momento en que la situación docente lo requiera, para que el futuro trabajador logre una mejor comprensión de los contenidos.

ABSTRACT Introduction: there is an enabling course designed for the training of comprehensive operators in surveillance and vector control. The teacher staff of Matanzas province has found some difficulties in the acquisition of knowledge by students. Objective: to characterize the surveillance and anti-vector labor force of the province of Matanzas, in order to design a set of indications that complement the contents of the course of enabling operators, so that students feel motivated and better understand the contents already included. Materials and methods: a descriptive, retrospective and longitudinal investigation was carried out from January to December 2019. Results: the group of operators is the most numerous. The educational level that prevailed was that of secondary school. In 2019, 17 training courses were conducted, in whose evaluations the teacher staff found difficulties in answering the questions of three of the course subjects, as well as in the use of the mother tongue. Conclusions: it was proposed to introduce new contents in each topic of the training course, which will be taught according to the forms of teaching and learning already defined in the programme. The teacher responsible for the subject will teach it at the moment when the teaching situation requires it, in order for the future worker to achieve a better understanding of the contents.

Startup and initial operation of an MLE-MABR treating municipal wastewater.

A 630 m3/d pilot plant was installed at Subiaco WRRF to determine design and operational parameters of a hybrid Modified Ludzack-Ettinger - Membrane Aerated Biofilm Reactor (MLE-MABR) configuration. Two commercial ZeeLung MABR cassettes were installed in series in the anoxic zone and the pilot was fed with primary effluent (averaging COD 601 mg/L, TKN 68.5 mg/L and 17-29 °C). A nitrifying biofilm was developed within 3 weeks and the nitrous oxide (N2O) gas emissions from the MABR exhaust gas proved to be a reliable parameter to assess biofilm development. Both MABRs achieved the average nitrification rate (NR) of 3.7 gNH4-N/m2.d when air flow was 8.6 and 11.2 Nm3/h to MABR1 and MABR2 respectively, which reached a maximum oxygen transfer rate of 17.4 gO2/m2.d. Biofilm thickness was controlled via air scouring and intermittent coarse bubble mixing (90 s on/90 s off). This paper discusses the startup strategy, minimum requirements for process monitoring, impact of different air flow conditions, ORP and mixing patterns on performance efficiency over a 22-week period.

Efectividad del temefos 1 % en larvas de Culex quinquefasciatus. Matanzas, 2019

RESUMEN Introducción: el temefos es el producto más utilizado para el tratamiento focal. Existe en diferentes formulaciones y se aplica para los depósitos de agua potable, en forma de gránulos de arena a una concentración del 1 %. Objetivo: determinar la duración de la efectividad del temefos, en su formulación costarricense Biolarv G1, en una población matancera de Culex quinquefasciatus. Materiales y métodos: se realizó un bioensayo de laboratorio, utilizando la F1 de una cepa matancera de Culex quinquefasciatus, la que se sometió a los efectos del Biolarv G1, lote 1180829. Se utilizaron tres variantes de recambio de agua potable. Resultados: la efectividad fue del 100 % de mortalidad larvaria hasta el 7º día en el recambio del 100 % de agua. Para el recambio del 50 % de agua, la mortalidad promedio del 1º al 17º día fue del 97,5 %. En el caso del recambio del 30 % de agua, la mortalidad fue del 100 % hasta el 27º día. Conclusiones: se demostró que el temefos, en su formulación Biolarv G1, puede tener mayor efectividad en recambios del 30 % de agua, con una durabilidad de alrededor de 30 días, lo que propicia la protección de los depósitos durante los ciclos de trabajo diseñados para la vigilancia y lucha antivectorial en Cuba.

ABSTRACT Introduction: temefos is the most widely used product for focal treatment. It exists in different formulations and is applied for drinking water tanks, in the form of sand-like granules at a concentration of 1 %. Objective: to determine the duration of temefos effectiveness, in its Costa Rican formulation Biolarv G1, in a Matanzas population of Culex quinquefasciatus. Materials and methods: a laboratory bioassay was performed, using the F1 of a Matanzas strain of Culex quinquefasciatus, which was subjected to the effects of Biolarv G1, lot 1180829. Three variants of drinking water replacement were used. Results: the effectiveness was 100% of larval mortality until the 7th day when 100 % of wáter was replaced. For the replacement of 50 % of water, average mortality was 97.5 % from the 1st to the 17th day. In the case of 30 % water replacement, mortality was 100 % up to the 27th day. Conclusions: it was shown that temefos, in its formulation Biolarv G1, can be more effective in replacements of 30 % of water, with a durability of about 30 days, which facilitates the protection of reservoirs during work cycles designed for surveillance and anti-vector control in Cuba.

Molecular Mechanisms in Pentanucleotide Repeat Diseases.

The number of neurodegenerative diseases resulting from repeat expansion has increased extraordinarily in recent years. In several of these pathologies, the repeat can be transcribed in RNA from both DNA strands producing, at least, one toxic RNA repeat that causes neurodegeneration by a complex mechanism. Recently, seven diseases have been found caused by a novel intronic pentanucleotide repeat in distinct genes encoding proteins highly expressed in the cerebellum. These disorders are clinically heterogeneous being characterized by impaired motor function, resulting from ataxia or epilepsy. The role that apparently normal proteins from these mutant genes play in these pathologies is not known. However, recent advances in previously known spinocerebellar ataxias originated by abnormal non-coding pentanucleotide repeats point to a gain of a toxic function by the pathogenic repeat-containing RNA that abnormally forms nuclear foci with RNA-binding proteins. In cells, RNA foci have been shown to be formed by phase separation. Moreover, the field of repeat expansions has lately achieved an extraordinary progress with the discovery that RNA repeats, polyglutamine, and polyalanine proteins are crucial for the formation of nuclear membraneless organelles by phase separation, which is perturbed when they are expanded. This review will cover the amazing advances on repeat diseases.

An audit of microplastic abundance throughout three Australian wastewater treatment plants.

Wastewater treatment plants (WWTPs) have been identified as an important pathway of microplastics to the environment. Most studies have focused on wastewater effluent, but generally only a small fraction of microplastics entering WWTPs are present in treated effluent. Instead, the majority of microplastics are expected to be retained in the sludge. To our knowledge, there is limited information on microplastics in sludge/biosolids from Australian WWTPs, despite 75% of biosolids produced in Australia being used for agriculture. This study evaluated the abundance of microplastics throughout the treatment trains of three WWTPs in Australia. The fate of microplastics >25 µm during treatment and their release to the environment was evaluated using an audit approach. The highest microplastic concentrations were detected in the influent, with fibres the dominant form of microplastic found. The screening and grit removal process preceding primary treatment removed 69-79% of microplastics, with these microplastics transported to landfill. Only 0.2-1.8% of the total microplastics in the influent were present in the final effluent, while 8-16% were retained in biosolids. This equates to between 22.1 × 106 to 133 × 106 microplastic particles per day released in effluent, between 864 × 106 to 1020 × 106 microplastic particles per day in biosolids, and between 4100 × 106 to 9100 × 106 microplastic particles per day transported to landfill. This study shows for the first time that most microplastics are retained during the initial screening and grit removal process with the load of microplastics going to landfill an order of magnitude greater than that in biosolids. Landfills may thus be an important sink (and potential future source) of microplastics from wastewater.

Joining European Scientific Forces to Face Pandemics.

Despite the international guidelines on the containment of the coronavirus disease 2019 (COVID-19) pandemic, the European scientific community was not sufficiently prepared to coordinate scientific efforts. To improve preparedness for future pandemics, we have initiated a network of nine European-funded Cooperation in Science and Technology (COST) Actions that can help facilitate inter-, multi-, and trans-disciplinary communication and collaboration.

Antisense Transcription across Nucleotide Repeat Expansions in Neurodegenerative and Neuromuscular Diseases: Progress and Mysteries.

Unstable repeat expansions and insertions cause more than 30 neurodegenerative and neuromuscular diseases. Remarkably, bidirectional transcription of repeat expansions has been identified in at least 14 of these diseases. More remarkably, a growing number of studies has been showing that both sense and antisense repeat RNAs are able to dysregulate important cellular pathways, contributing together to the observed clinical phenotype. Notably, antisense repeat RNAs from spinocerebellar ataxia type 7, myotonic dystrophy type 1, Huntington's disease and frontotemporal dementia/amyotrophic lateral sclerosis associated genes have been implicated in transcriptional regulation of sense gene expression, acting either at a transcriptional or posttranscriptional level. The recent evidence that antisense repeat RNAs could modulate gene expression broadens our understanding of the pathogenic pathways and adds more complexity to the development of therapeutic strategies for these disorders. In this review, we cover the amazing progress made in the understanding of the pathogenic mechanisms associated with repeat expansion neurodegenerative and neuromuscular diseases with a focus on the impact of antisense repeat transcription in the development of efficient therapies.

Mutational mechanism for DAB1 (ATTTC)n insertion in SCA37: ATTTT repeat lengthening and nucleotide substitution.

Dynamic mutations by microsatellite instability are the molecular basis of a growing number of neuromuscular and neurodegenerative diseases. Repetitive stretches in the human genome may drive pathogenicity, either by expansion above a given threshold, or by insertion of abnormal tracts in nonpathogenic polymorphic repetitive regions, as is the case in spinocerebellar ataxia type 37 (SCA37). We have recently established that this neurodegenerative disease is caused by an (ATTTC)n insertion within an (ATTTT)n in a noncoding region of DAB1. We now investigated the mutational mechanism that originated the (ATTTC)n insertion within an ancestral (ATTTT)n . Approximately 3% of nonpathogenic (ATTTT)n alleles are interspersed by AT-rich motifs, contrarily to mutant alleles that are composed of pure (ATTTT)n and (ATTTC)n stretches. Haplotype studies in unaffected chromosomes suggested that the primary mutational mechanism, leading to the (ATTTC)n insertion, was likely one or more T>C substitutions in an (ATTTT)n pure allele of approximately 200 repeats. Then, the (ATTTC)n expanded in size, originating a deleterious allele in DAB1 that leads to SCA37. This is likely the mutational mechanism in three similar (TTTCA)n insertions responsible for familial myoclonic epilepsy. Because (ATTTT)n tracts are frequent in the human genome, many loci could be at risk for this mutational process.

A repeat-primed PCR assay for pentanucleotide repeat alleles in spinocerebellar ataxia type 37.

Spinocerebellar ataxia 37 (SCA37) is caused by an (ATTTC)n insertion in a polymorphic ATTTT repeat in the non-coding region of DAB1. The non-pathogenic alleles have a configuration [(ATTTT)7-400], whereas pathogenic alleles have a complex structure of [(ATTTT)60-79(ATTTC)31-75(ATTTT)58-90]. Molecular diagnosis of SCA37 is laborious because about 7% of the pentanucleotide repeat alleles in DAB1 are larger than 30 units and, thus, fail to amplify with standard PCR conditions, resulting in apparently homoallelism or in complete lack of PCR amplification in several cases. The molecular test currently available requires long-range PCR and sequencing analysis for the detection and characterization of these large alleles. We developed a simple assay capable of rapidly detecting the presence or absence of large pentanucleotide repeat sizes. This assay is based on repeat-primed PCR followed by high-throughput capillary electrophoresis. Combining the standard PCR with RP-PCR allows completion of the diagnosis in more than 80% of individuals, minimizing the number of samples that require long-range PCR followed by Sanger sequencing analysis. This assay meets many of the requirements for pre-screening of large cohorts of affected individuals.

Vigilancia entomológica de culícidos en la provincia de Matanzas durante el año 2015 / Entomological surveillance of culicids in the province of Matanzas during 2015

Introducción: la vigilancia entomológica es la recolección de información sobre los vectores de enfermedades y su entorno. Es un componente de la vigilancia epidemiológica, que consiste en una actividad del control de los vectores que se realiza durante todo el año, para conocer la presencia de estos en un área geográfica, la densidad de sus poblaciones, así como identificar los principales criaderos y los más productivos. Objetivo: identificación de las especies de culícidos que circularon en la provincia de Matanzas, durante el año 2015, así como la definición de la especie que predominó y tipificación de los depósitos más colonizados por estos en su fase de vida inmadura. Materiales y Métodos: se realizó un análisis de las estadísticas de la vigilancia entomológica durante el año 2015. Según la metodología descrita en el Programa Nacional de Vigilancia y Lucha Antivectorial se clasificó los depósitos, se colectaron y diagnosticaron las muestras. Resultados: las muestras larvarias aportaron la mayor cantidad de datos. Los depósitos de agua situados en el exterior de las viviendas, y dentro de estos las larvitrampas fueron los más utilizados por los mosquitos. La especie predominante en fase inmadura fue Aedes albopictus, mientras que en fase adulta Culex quinquefasciatus. Conclusiones: se identificaron 5 géneros con 25 especies de culícidos, el 36,8 % de la fauna cubana. Como especie predominante se define al A. albopictus. Los depósitos más explotados por estos organismos según su ubicación, fueron los del exterior, y según su tipo, fueron las larvitrampas (grupo E) (AU).

Introduction: the entomological surveillance is the recollection of information on diseases´ vectors and their environment. It is part of the epidemiologic surveillance, and it is an activity of vectors controlling carried out around the year to determine their presence in a geographical area, the density of their populations, and to identify their main breeding places and the most productive ones. Objectives: to identify the culicids species living in the province of Matanzas during 2015, and also to define the predominating specie and to typify the deposits more colonized by them in the in-mature life stage. Materials and Methods: it was carried out an analysis of the entomological surveillance statistics during 2015. The reservoirs were classified, and the samples were collected and diagnosed according to the methodology described in the National Program of Antivectorial Surveillance and Struggle. Results: the larval samples gave the biggest quantity of data. The water reservoirs located outside the living accommodations, and among them the larval traps were the most used by mosquitoes. The predominant specie found in in-mature stage was Aedes albopictus, while Culex quinquefasciatus predominated in adult stage. Conclusions: 5 genus with 25 species of culicids were found, 36.8 of them part of the Cuban fauna. The Aedes albopictus was defined as the predominant specie. The reservoirs most used by these organisms were, according to their location, those located outside, and according to their type, the larval traps (group E) (AU).

Vigilancia entomológica de culícidos en la provincia de Matanzas durante el año 2015 / Entomological surveillance of culicids in the province of Matanzas during 2015

Introducción: la vigilancia entomológica es la recolección de información sobre los vectores de enfermedades y su entorno. Es un componente de la vigilancia epidemiológica, que consiste en una actividad del control de los vectores que se realiza durante todo el año, para conocer la presencia de estos en un área geográfica, la densidad de sus poblaciones, así como identificar los principales criaderos y los más productivos. Objetivo: identificación de las especies de culícidos que circularon en la provincia de Matanzas, durante el año 2015, así como la definición de la especie que predominó y tipificación de los depósitos más colonizados por estos en su fase de vida inmadura. Materiales y Métodos: se realizó un análisis de las estadísticas de la vigilancia entomológica durante el año 2015. Según la metodología descrita en el Programa Nacional de Vigilancia y Lucha Antivectorial se clasificó los depósitos, se colectaron y diagnosticaron las muestras. Resultados: las muestras larvarias aportaron la mayor cantidad de datos. Los depósitos de agua situados en el exterior de las viviendas, y dentro de estos las larvitrampas fueron los más utilizados por los mosquitos. La especie predominante en fase inmadura fue Aedes albopictus, mientras que en fase adulta Culex quinquefasciatus. Conclusiones: se identificaron 5 géneros con 25 especies de culícidos, el 36,8 % de la fauna cubana. Como especie predominante se define al A. albopictus. Los depósitos más explotados por estos organismos según su ubicación, fueron los del exterior, y según su tipo, fueron las larvitrampas (grupo E) (AU).

Introduction: the entomological surveillance is the recollection of information on diseases´ vectors and their environment. It is part of the epidemiologic surveillance, and it is an activity of vectors controlling carried out around the year to determine their presence in a geographical area, the density of their populations, and to identify their main breeding places and the most productive ones. Objectives: to identify the culicids species living in the province of Matanzas during 2015, and also to define the predominating specie and to typify the deposits more colonized by them in the in-mature life stage. Materials and Methods: it was carried out an analysis of the entomological surveillance statistics during 2015. The reservoirs were classified, and the samples were collected and diagnosed according to the methodology described in the National Program of Antivectorial Surveillance and Struggle. Results: the larval samples gave the biggest quantity of data. The water reservoirs located outside the living accommodations, and among them the larval traps were the most used by mosquitoes. The predominant specie found in in-mature stage was Aedes albopictus, while Culex quinquefasciatus predominated in adult stage. Conclusions: 5 genus with 25 species of culicids were found, 36.8 of them part of the Cuban fauna. The Aedes albopictus was defined as the predominant specie. The reservoirs most used by these organisms were, according to their location, those located outside, and according to their type, the larval traps (group E) (AU).

A Pentanucleotide ATTTC Repeat Insertion in the Non-coding Region of DAB1, Mapping to SCA37, Causes Spinocerebellar Ataxia.

Advances in human genetics in recent years have largely been driven by next-generation sequencing (NGS); however, the discovery of disease-related gene mutations has been biased toward the exome because the large and very repetitive regions that characterize the non-coding genome remain difficult to reach by that technology. For autosomal-dominant spinocerebellar ataxias (SCAs), 28 genes have been identified, but only five SCAs originate from non-coding mutations. Over half of SCA-affected families, however, remain without a genetic diagnosis. We used genome-wide linkage analysis, NGS, and repeat analysis to identify an (ATTTC)n insertion in a polymorphic ATTTT repeat in DAB1 in chromosomal region 1p32.2 as the cause of autosomal-dominant SCA; this region has been previously linked to SCA37. The non-pathogenic and pathogenic alleles have the configurations [(ATTTT)7-400] and [(ATTTT)60-79(ATTTC)31-75(ATTTT)58-90], respectively. (ATTTC)n insertions are present on a distinct haplotype and show an inverse correlation between size and age of onset. In the DAB1-oriented strand, (ATTTC)n is located in 5' UTR introns of cerebellar-specific transcripts arising mostly during human fetal brain development from the usage of alternative promoters, but it is maintained in the adult cerebellum. Overexpression of the transfected (ATTTC)58 insertion, but not (ATTTT)n, leads to abnormal nuclear RNA accumulation. Zebrafish embryos injected with RNA of the (AUUUC)58 insertion, but not (AUUUU)n, showed lethal developmental malformations. Together, these results establish an unstable repeat insertion in DAB1 as a cause of cerebellar degeneration; on the basis of the genetic and phenotypic evidence, we propose this mutation as the molecular basis for SCA37.

Characterization of polymeric nanoparticles for intravenous delivery: Focus on stability.

The nano-bio interaction has been of increased focus in the past years but very limited results have been obtained for polymeric nanoparticles (NP). Not only is needed to broaden the results obtained with model NP towards other nano-materials used for clinical application but the colloidal stability of NP as a variable consequence of the formation of the protein corona has been significantly understated. The lack and heterogeneity of assays to study NP stability and represent the biological environment call for the standardization of assays to improve the representativeness and comparability of results. In this paper, uncoated and PAH-coated PLGA NP have been prepared and characterized in regard to their potential for intravenous administration. The comparative study of the stability of NP in three media used to represent the biological environment-bovine serum albumin (BSA) solution, mouse and human plasma - revealed that both formulations were unstable in human plasma as opposed to the results obtained for other media. This unexpected behavior in plasmas of different origins could be correlated with a significant variation of the amount of proteins adsorbed to NP and, ultimately, with an approximately 6-fold difference in total protein concentration between the plasma samples. These results suggest that inter-species variation could impact on the colloidal stability of NP and enhance the need to understand the correlation between biological media and identify protocol-related interferences which, altogether, may evidence a relevant factor compromising in vitro- in vivo correlation and the translation of delivery systems aimed at intravenous administration.

Recent Advances in Nucleic Acid-Based Delivery: From Bench to Clinical Trials in Genetic Diseases.

Delivery of nucleic acids is the most promising therapy for many diseases that remain untreatable. Therefore, many research efforts have been put on finding a safe and efficient delivery system able to provide a sustained response. Viral vectors have proved to be the most efficient for delivery of nucleic acids and, thus, stand as the foremost vector used in current clinical trials. However, safety issues arise as a main concern and mitigate their use, impelling the improvement of non-viral alternatives. This review focuses on the recent advances in pre-clinical development of non-viral polyplexes and lipoplexes for nucleic acid-based delivery, in contrast with vectors being used in present clinical trials. Nucleic acid vectors for neurodegenerative ataxias, Parkinson's disease, retinitis pigmentosa, cystic fibrosis, hemophilia, pancreatic and lung cancer, and rheumatoid arthritis are discussed to illustrate current state of pre-clinical and clinical studies. Thereby, denoting the prospects for treatment of genetic diseases and elucidating the trend in non-viral vector development and improvement which is expected to significantly increase disease rescue exceeding the modest clinical successes observed so far.

Unstable repeat expansions in neurodegenerative diseases: nucleocytoplasmic transport emerges on the scene.

An astonishing number of neurological diseases result from expansion of unstable repetitive sequences causing alterations in key neuronal processes. Some are progressive late-onset conditions related to aging, such as the spinocerebellar ataxias. In several of these pathologies, the expanded repeat is transcribed, producing an expanded RNA repeat that causes neurodegeneration by a complex mechanism, comprising 3 main pathways. These include (1) accumulation in the nucleus of RNA foci, resulting from sequestration of RNA-binding proteins functioning in important neuronal cascades; (2) decrease in availability of RNA-binding proteins, such as splicing factors, causing alternative splicing misregulation with imbalance in the expression ratio of neuronal isoforms; and (3) generation of neurotoxic peptides, produced from repeat-associated non-ATG-initiated translation across the RNA repeat, in all reading frames. Recently, 2 pathologies characterized by impaired motor function, cognitive decline, or/and degeneration of motor neurons have been found that have broaden our understanding of these diseases. Moreover, the finding of compromised nucleocytoplasmic transport opens new avenues for research. This review will cover the amazing progress regarding these conditions.

Recent advances in characterization of nonviral vectors for delivery of nucleic acids: impact on their biological performance.

INTRODUCTION: Nucleic acid delivery is a complex process that requires transport across numerous extracellular and intracellular barriers, whose impact is often neglected during optimization studies. As such, the development of nonviral vectors for efficient delivery would benefit from an understanding of how these barriers relate to the physicochemical properties of lipoplexes and polyplexes. AREAS COVERED: This review focuses on the evaluation of parameters associated with barriers to delivery such as blood and immune cells compatibility which, as a collective, may serve as a useful prescreening tool for the advancement of nonviral vectors in vivo. An outline of the most relevant rationally developed polyplexes and lipoplexes for clinical application is also given. EXPERT OPINION: The evaluation of scientifically recognized parameters enabled the identification of systemic delivered nonviral vectors' behavior while in blood as one of the key determinants of vectors function and activity both in vitro and in vivo. This multiparametric approach complements the use of in vitro efficacy results alone for prescreening and improves in vitro-in vivo translation by minimizing false negatives. Further, it can aid in the identification of meaningful structure-function-activity relationships, improve the in vitro screening process of nonviral vectors before in vivo use and facilitate the future development of potent and safe nonviral vectors.