Gene and cell therapy fields have experienced remarkable growth during the past decade. Demands for preclinical and clinical safety assessments of these cell and gene therapy test articles (TAs) have ...effectively increased the necessity for regulated biodistribution, vector shedding, gene expression, and/or pharmacokinetics bioanalysis studies. Guidance documents issued from numerous international regulatory authorities recommend the use of quantitative polymerase chain reaction (qPCR) and/or quantitative reverse transcriptase PCR (qRT-PCR) assays due to their highly sensitive and robust target-specific detection. However, only preclinical biodistribution assay sensitivity is specified in these documents. Criteria such as accuracy, precision, and repeatability are not yet defined. This guidance void has resulted in several conflicting institutional interpretations of essential parameters necessary for the development and validation of robust assays to support safety assessments of gene and cell therapy TAs. There is an urgent need for an ongoing discussion among bioanalytical scientists in this field to generate a “best practice” consensus around preclinical and clinical qPCR/qRT-PCR assay design. With regard to this need, we offer critical points to consider when developing, validating, running sample analysis, and reporting qPCR/qRT-PCR assays.
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Ma and colleagues offer critical points to consider when developing and validating qPCR/qRT-PCR assays and analyzing the biodistribution and vector shedding samples to support the preclinical and clinical safety assessments of the gene and cell therapy test articles. Assay acceptance criteria are recommended to the scientific community.
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GEOZS, IJS, IMTLJ, KILJ, KISLJ, NLZOH, NUK, OILJ, PNG, SAZU, SBCE, SBJE, UILJ, UL, UM, UPCLJ, UPUK, ZAGLJ, ZRSKP
We present a scanning magnetic force sensor based on an individual magnet-tipped GaAs nanowire (NW) grown by molecular beam epitaxy. Its magnetic tip consists of a final segment of single-crystal ...MnAs formed by sequential crystallization of the liquid Ga catalyst droplet. We characterize the mechanical and magnetic properties of such NWs by measuring their flexural mechanical response in an applied magnetic field. Comparison with numerical simulations allows the identification of their equilibrium magnetization configurations, which in some cases include magnetic vortices. To determine a NW’s performance as a magnetic scanning probe, we measure its response to the field profile of a lithographically patterned current-carrying wire. The NWs’ tiny tips and their high force sensitivity make them promising for imaging weak magnetic field patterns on the nanometer-scale, as required for mapping mesoscopic transport and spin textures or in nanometer-scale magnetic resonance.
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IJS, KILJ, NUK, PNG, UL, UM
Abstract
Summary
Whole genome assembly (WGA) of bacterial genomes with short reads is a quite common task as DNA sequencing has become cheaper with the advances of its technology. The process of ...assembling a genome has no absolute golden standard and it requires to perform a sequence of steps each of which can involve combinations of many different tools. However, the quality of the final assembly is always strongly related to the quality of the input data. With this in mind we built WGA-LP, a package that connects state-of-the-art programs for microbial analysis and novel scripts to check and improve the quality of both samples and resulting assemblies. WGA-LP, with its conservative decontamination approach, has shown to be capable of creating high quality assemblies even in the case of contaminated reads.
Availability and implementation
WGA-LP is available on GitHub (https://github.com/redsnic/WGA-LP) and Docker Hub (https://hub.docker.com/r/redsnic/wgalp). The web app for node visualization is hosted by shinyapps.io (https://redsnic.shinyapps.io/ContigCoverageVisualizer/).
Supplementary information
Supplementary data are available at Bioinformatics online.
Adenosine diphosphate (ADP)‐ribosylation is a post‐translational protein modification implicated in the regulation of a range of cellular processes. A family of proteins that catalyse ...ADP‐ribosylation reactions are the poly(ADP‐ribose) (PAR) polymerases (PARPs). PARPs covalently attach an ADP‐ribose nucleotide to target proteins and some PARP family members can subsequently add additional ADP‐ribose units to generate a PAR chain. The hydrolysis of PAR chains is catalysed by PAR glycohydrolase (PARG). PARG is unable to cleave the mono(ADP‐ribose) unit directly linked to the protein and although the enzymatic activity that catalyses this reaction has been detected in mammalian cell extracts, the protein(s) responsible remain unknown. Here, we report the homozygous mutation of the c6orf130 gene in patients with severe neurodegeneration, and identify C6orf130 as a PARP‐interacting protein that removes mono(ADP‐ribosyl)ation on glutamate amino acid residues in PARP‐modified proteins. X‐ray structures and biochemical analysis of C6orf130 suggest a mechanism of catalytic reversal involving a transient C6orf130 lysyl‐(ADP‐ribose) intermediate. Furthermore, depletion of C6orf130 protein in cells leads to proliferation and DNA repair defects. Collectively, our data suggest that C6orf130 enzymatic activity has a role in the turnover and recycling of protein ADP‐ribosylation, and we have implicated the importance of this protein in supporting normal cellular function in humans.
Crystal structure and biochemical data reveal a gene mutated in patients with severe neurodegeneration to encode an elusive enzyme for removing ADP‐ribose from proteins.
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FZAB, GIS, IJS, KILJ, NLZOH, NUK, OILJ, SBCE, SBMB, UL, UM, UPUK
The last decade has seen the development of a growing tendency to use the most modern technologies, in the field of Cultural Heritage, with the aim of digitizing and facilitating protection and ...conservation activities. Much research has focused on the development of innovative methods such as photogrammetry or Terrestrial Laser Scanners, in terms of reliability, precision, time and costs. In this research, however, the use of the smartphone was investigated by comparing the point clouds obtained via videogrammetry from smartphones, with those generated by different digital survey techniques, such as Terrestrial Laser Scanners and photogrammetry via SLR camera. Specifically, a smartphone was used and the comparison between point clouds was conducted based on four criteria: point clouds fitting, density evaluation, profiling and texture quality with the aim, therefore, of verifying the geometric reliability of the data and the quality of polygonal lines and the mesh/texture derived. The case study selected was the sepulchral monument of the Pascopepe Lambertini family (14th century), located in the Crypt of Santa Maria della Scala, the Cathedral of Trani (South of Italy). Finally, the research demonstrates how this methodology, in the documentation of heritage, allows for even greater portability and accessibility compared to other methodologies, maintaining widely acceptable standards of accuracy and therefore going to constitute a valid alternative in the documentation of historical heritage.
We use a scanning nanometer-scale superconducting quantum interference device to map the stray magnetic field produced by individual ferromagnetic nanotubes (FNTs) as a function of applied magnetic ...field. The images are taken as each FNT is led through magnetic reversal and are compared with micromagnetic simulations, which correspond to specific magnetization configurations. In magnetic fields applied perpendicular to the FNT long axis, their magnetization appears to reverse through vortex states, that is, configurations with vortex end domains or in the case of a sufficiently short FNT with a single global vortex. Geometrical imperfections in the samples and the resulting distortion of idealized magnetization configurations influence the measured stray-field patterns.
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IJS, KILJ, NUK, PNG, UL, UM
Aim of this study was to investigate the activation of the IFNγ pathway in the affected liver and in the blood of patients with secondary hemophagocytic lymphohistiocytosis (sHLH). To this purpose, ...the mRNA expression levels of IFNG and IFNγ-inducible genes as well as Tyrosine (701)-phosphorylated signal transducer and activator of transcription 1 (STAT1) protein levels were evaluated in the liver and in peripheral blood mononuclear cells (PBMCs) of three patients with sHLH with predominant liver involvement. The mRNA expression levels of IFNG and IFNγ-inducible genes were markedly higher in patient livers compared to control livers and to one disease control liver. Conversely, slight differences in the expression levels of Type I IFN-inducible genes and other classical inflammatory cytokine genes were found. Further supporting the activation of the IFNγ pathway, higher protein levels of phosphorylated and total STAT1 were detected in patient livers compared to control livers. When the expression of the same genes analysed in liver tissues was evaluated in PBMCs collected from 2 out of 3 patients before the liver biopsy, we found that mRNA levels of IFNγ-inducible genes were markedly increased. Accordingly, high circulating levels of IFNγ-inducible CXCL9 were observed in patients. Altogether, these data demonstrate the selective and marked up-regulation of the IFNγ pathway in the liver tissue and blood of patients with active sHLH. Finally, we show that measurement of circulating CXCL9 levels and evaluation of IFNγ-inducible gene expression levels in PBMCs may represent a new valid tool to better identify patients with suspected HLH with predominant liver involvement.
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DOBA, IZUM, KILJ, NUK, PILJ, PNG, SAZU, SIK, UILJ, UKNU, UL, UM, UPUK
Poly(ADP-ribose)polymerase-1 (PARP-1) is a predominantly nuclear enzyme that exerts numerous functions in cellular physiology and pathology, from maintenance of DNA stability to transcriptional ...regulation. Through a proteomic analysis of PARP-1 co-immunoprecipitation complexes, we identified Mitofilin, a mitochondrial protein, as a new PARP-1 interactor. This result prompted us to further investigate the presence and the role of the enzyme in mitochondria. Using laser confocal microscopy and Western blot analysis of purified mitochondria, we demonstrated the mitochondrial localization of a fraction of PARP-1. Further, the effects of overexpressing or down-regulating Mitofilin showed that this protein promotes and is required for PARP-1 mitochondrial localization. We also report several lines of evidence suggesting that intramitochondrial PARP-1 plays a role in mitochondrial DNA (mtDNA) damage signaling and/or repair. First, we show that PARP-1 binds to different regions throughout the mtDNA. Moreover, we demonstrated that the depletion of either PARP-1 or Mitofilin, which abrogates the mitochondrial localization of the enzyme, leads to the accumulation of mtDNA damage. Finally, we show that DNA ligase III, known to be required for mtDNA repair, participates in a PARP-1-containing complex bound to mtDNA. This work highlights a new environment for PARP-1, opening the possibility that at least some of the nuclear functions of the enzyme can be also extended to mtDNA metabolism.
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GEOZS, IJS, IMTLJ, KILJ, KISLJ, NLZOH, NUK, OILJ, PNG, SAZU, SBCE, SBJE, UILJ, UL, UM, UPCLJ, UPUK, ZAGLJ, ZRSKP
Background
Williams syndrome (WS) phenotype is described as unique and intriguing. The aim of this study was to investigate the associations between speech–language abilities, general cognitive ...functioning and behavioural problems in individuals with WS, considering age effects and speech–language characteristics of WS sub‐groups.
Methods
The study's participants were 26 individuals with WS and their parents. General cognitive functioning was assessed with the Wechsler Intelligence Scale. Peabody Picture Vocabulary Test, Token Test and the Cookie Theft Picture test were used as speech–language measures. Five speech–language characteristics were evaluated from a 30‐min conversation (clichés, echolalia, perseverative speech, exaggerated prosody and monotone intonation). The Child Behaviour Checklist (CBCL 6‐18) was used to assess behavioural problems.
Results
Higher single‐word receptive vocabulary and narrative vocabulary were negatively associated with CBCL T‐scores for Social Problems, Aggressive Behaviour and Total Problems. Speech rate was negatively associated with the CBCL Withdrawn/Depressed T‐score. Monotone intonation was associated with shy behaviour, as well as exaggerated prosody with talkative behaviour. WS with perseverative speech and exaggerated prosody presented higher scores on Thought Problems. Echolalia was significantly associated with lower Verbal IQ. No significant association was found between IQ and behaviour problems. Age‐associated effects were observed only for the Aggressive Behaviour scale.
Conclusions
Associations reported in the present study may represent an insightful background for future predictive studies of speech–language, cognition and behaviour problems in WS.
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BFBNIB, DOBA, FZAB, GIS, IJS, IZUM, KILJ, NLZOH, NUK, OILJ, PILJ, PNG, SAZU, SBCE, SBMB, SIK, UILJ, UKNU, UL, UM, UPUK, VSZLJ
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