Improving CRISPR/Cas9 mutagenesis efficiency by delaying the early development of zebrafish embryos

CRISPR/Cas9 driven mutagenesis in zygotes is a popular tool for introducing targeted mutations in model organisms. Compared to mouse, mutagenesis in zebrafish is relatively inefficient and results in somatic mosaicism most likely due to a short single-cell stage of about 40 min. Here we explored two options to improve CRISPR/Cas9 mutagenesis in zebrafish-extending the single-cell stage and defining conditions for carrying out mutagenesis in oocytes prior to in vitro fertilization. Previous work has shown that ovarian fluid from North American salmon species (coho and chinook salmon) prolong oocyte survival ex vivo so that they are viable for hours instead of dying within minutes if left untreated. We found that commonly farmed rainbow trout (Oncorhynchus mykiss) ovarian fluid (RTOF) has similar effect on zebrafish oocyte viability. In order to prolong single-cell stage, we incubated zebrafish zygotes in hydrogen sulfide (H2S) and RTOF but failed to see any effect. However, the reduction of temperature from standard 28 to 12 degrees C postponed the first cell division by about an hour. In addition, the reduction in temperature was associated with increased CRISPR/Cas9 mutagenesis rate. These results suggest that the easily applicable reduction in temperature facilitates CRISPR/Cas9 mutagenesis in zebrafish.Peer reviewe

Gaussian mixture models and machine learning predict megakaryocytic growth and differentiation potential ex vivo

The ability to analyze single cells via flow cytometry has resulted in a wide range of biological and medical applications. Currently, there is no established framework to compare and interpret time-series flow cytometry data for cell engineering applications. Manual analysis of temporal trends is time-consuming and subjective for large-scale datasets. We resolved this bottleneck by developing TEmporal Gaussian Mixture models (TEGM), an unbiased computational strategy to quantify and predict temporal trends of developing cell subpopulations indicative of cellular phenotype. TEGM applies Gaussian mixture models and gradient boosted trees for cell engineering applications. TEGM enables the extraction of subtle features, such as the dispersion and rate of change of surface marker expression for each subpopulation over time. These critical, yet hard-to-discern, features are fed into machine-learning algorithms that predict underlying cell classes. Our framework can be flexibly applied to conventional flow cytometry sampling schemes, and allows for faster and more consistent processing of time-series flow cytometry data. Please click Additional Files below to see the full abstract

Using Gaussian mixture models and machine learning to predict donor- dependent megakaryocytic cell growth and differentiation potential ex vivo

The ability to analyze single cells via flow cytometry has resulted in a wide range of biological and medical applications. Currently, there is no established framework to compare and interpret time-series flow cytometry data for cell engineering applications. Manual analysis of temporal trends is time-consuming and subjective for large-scale datasets. We resolved this bottleneck by developing TEmporal Gaussian Mixture models (TEGM), an unbiased computational strategy to quantify and predict temporal trends of developing cell subpopulations indicative of cellular phenotype.. Please click Additional Files below to see the full abstract

Defining alternative recovery strategies for reuse: An analysis of multiple case studies under the reuse umbrella

This study focuses on the role of the reuse recovery approach and its strategies in the circular economy through a product design lens. While the circular economy has been widely discussed, not all recovery strategies have been thoroughly investigated and understood alike. This research differentiates between reuse and other recovery strategies and defines three distinct opportunities under the ‘reuse umbrella’ – reuse, cascade and relink. For each opportunity, possible sub-opportunities are identified and elaborated. Through a multiple case study analysis, the sub-opportunities are elaborated with a "Design for X" approach into eight different strategies of reuse, including design for direct reuse, reconfiguration, material cascading, repurposing, component reuse, creative reuse, material reclamation and adaptive reuse. The research emphasises that the appropriateness of a reuse strategy depends on various aspects such as the context, users, industries, and material flows. Also, this study stresses the importance of reuse in promoting circular product development and consumption, providing valuable implications for businesses and designers.The research project received financial backing from the UKRI National Interdisciplinary Circular Economy Research programme (NICER

(E)-N′-(2-Hydroxy­benzyl­idene)-2-(4-isobutyl­phen­yl)propanohydrazide

The title hydrazide compound, C20H24N2O2, exists in a trans configuration with respect to the acyclic C=N bond and an intra­molecular O—H⋯N hydrogen bond generates an S(6) ring motif. The mean plane through the formohydrazide unit is essentially planar [maximum deviation = 0.025 (2) Å], and forms dihedral angles of 24.45 (16) and 87.14 (16)° with the two benzene rings. In the crystal structure, inter­molecular N—H⋯O and C—H⋯O hydrogen bonds link neighbouring mol­ecules into extended chains along the c axis, which incorporate R 2 2(16) ring motifs. An inter­molecular C—H⋯π inter­action is also observed

TRAIL Death Receptor-4, Decoy Receptor-1 and Decoy Receptor-2 Expression on CD8+ T Cells Correlate with the Disease Severity in Patients with Rheumatoid Arthritis

BACKGROUND: Rheumatoid Arthritis (RA) is a chronic autoimmune inflammatory disorder. Although the pathogenesis of disease is unclear, it is well known that T cells play a major role in both development and perpetuation of RA through activating macrophages and B cells. Since the lack of TNF-Related Apoptosis Inducing Ligand (TRAIL) expression resulted in defective thymocyte apoptosis leading to an autoimmune disease, we explored evidence for alterations in TRAIL/TRAIL receptor expression on peripheral T lymphocytes in the molecular mechanism of RA development. METHODS: The expression of TRAIL/TRAIL receptors on T cells in 20 RA patients and 12 control individuals were analyzed using flow cytometry. The correlation of TRAIL and its receptor expression profile was compared with clinical RA parameters (RA activity scored as per DAS28) using Spearman Rho Analysis. RESULTS: While no change was detected in the ratio of CD4+ to CD8+ T cells between controls and RA patient groups, upregulation of TRAIL and its receptors (both death and decoy) was detected on both CD4+ and CD8+ T cells in RA patients compared to control individuals. Death Receptor-4 (DR4) and the decoy receptors DcR1 and DcR2 on CD8+ T cells, but not on CD4+ T cells, were positively correlated with patients' DAS scores. CONCLUSIONS: Our data suggest that TRAIL/TRAIL receptor expression profiles on T cells might be important in revelation of RA pathogenesis

Ret is essential to mediate GDNF’s neuroprotective and neuroregenerative effect in a Parkinson disease mouse model

Glial cell line-derived neurotrophic factor (GDNF) is a potent survival and regeneration-promoting factor for dopaminergic neurons in cell and animal models of Parkinson disease (PD). GDNF is currently tested in clinical trials on PD patients with so far inconclusive results. The receptor tyrosine kinase Ret is the canonical GDNF receptor, but several alternative GDNF receptors have been proposed, raising the question of which signaling receptor mediates here the beneficial GDNF effects. To address this question we overexpressed GDNF in the striatum of mice deficient for Ret in dopaminergic neurons and subsequently challenged these mice with 1-methyl-4-phenyl-1,2,3,6-tetrahydropyridine (MPTP). Strikingly, in this established PD mouse model, the absence of Ret completely abolished GDNF’s neuroprotective and regenerative effect on the midbrain dopaminergic system. This establishes Ret signaling as absolutely required for GDNF’s effects to prevent and compensate dopaminergic system degeneration and suggests Ret activation as the primary target of GDNF therapy in PD

NRF2 Activation Restores Disease Related Metabolic Deficiencies in Olfactory Neurosphere-Derived Cells from Patients with Sporadic Parkinson's Disease

Extent: 14p.Background: Without appropriate cellular models the etiology of idiopathic Parkinson’s disease remains unknown. We recently reported a novel patient-derived cellular model generated from biopsies of the olfactory mucosa (termed olfactory neurosphere-derived (hONS) cells) which express functional and genetic differences in a disease-specific manner. Transcriptomic analysis of Patient and Control hONS cells identified the NRF2 transcription factor signalling pathway as the most differentially expressed in Parkinson’s disease. Results: We tested the robustness of our initial findings by including additional cell lines and confirmed that hONS cells from Patients had 20% reductions in reduced glutathione levels and MTS [3-(4,5-dimethylthiazol-2-yl)-5-(3-carboxymethoxyphenyl)- 2-(4-sulfophenyl)-2H-tetrazolium, inner salt] metabolism compared to cultures from healthy Control donors. We also confirmed that Patient hONS cells are in a state of oxidative stress due to higher production of H2O2 than Control cultures. siRNA-mediated ablation of NRF2 in Control donor cells decreased both total glutathione content and MTS metabolism to levels detected in cells from Parkinson’s Disease patients. Conversely, and more importantly, we showed that activation of the NRF2 pathway in Parkinson’s disease hONS cultures restored glutathione levels and MTS metabolism to Control levels. Paradoxically, transcriptomic analysis after NRF2 pathway activation revealed an increased number of differentially expressed mRNAs within the NRF2 pathway in L-SUL treated Patient-derived hONS cells compared to L-SUL treated Controls, even though their metabolism was restored to normal. We also identified differential expression of the PI3K/AKT signalling pathway, but only post-treatment. Conclusions: Our results confirmed NRF2 as a potential therapeutic target for Parkinson’s disease and provided the first demonstration that NRF2 function was inducible in Patient-derived cells from donors with uniquely varied genetic backgrounds. However, our results also demonstrated that the response of PD patient-derived cells was not co-ordinated in the same way as in Control cells. This may be an important factor when developing new therapeutics.Anthony L. Cook, Alejandra M. Vitale, Sugandha Ravishankar, Nicholas Matigian, Greg T. Sutherland, Jiangou Shan, Ratneswary Sutharsan, Chris Perry, Peter A. Silburn, George D. Mellick, Murray L. Whitelaw, Christine A. Wells, Alan Mackay-Sim and Stephen A. Woo

The Neurotoxicity of DOPAL: Behavioral and Stereological Evidence for Its Role in Parkinson Disease Pathogenesis

BACKGROUND: The etiology of Parkinson disease (PD) has yet to be fully elucidated. We examined the consequences of injections of 3,4-dihydroxyphenylacetaldehyde (DOPAL), a toxic metabolite of dopamine, into the substantia nigra of rats on motor behavior and neuronal survival. METHODS/PRINCIPAL FINDINGS: A total of 800 nl/rat of DOPAL (1 µg/200 nl) was injected stereotaxically into the substantia nigra over three sites while control animals received similar injections of phosphate buffered saline. Rotational behavior of these rats was analyzed, optical density of striatal tyrosine hydroxylase was calculated, and unbiased stereological counts of the substantia nigra were made. The rats showed significant rotational asymmetry ipsilateral to the lesion, supporting disruption of dopaminergic nigrostriatal projections. Such disruption was verified since the density of striatal tyrosine hydroxylase decreased significantly (p<0.001) on the side ipsilateral to the DOPAL injections when compared to the non-injected side. Stereological counts of neurons stained for Nissl in pars compacta of the substantia nigra significantly decreased (p<0.001) from control values, while counts of those in pars reticulata were unchanged after DOPAL injections. Counts of neurons immunostained for tyrosine hydroxylase also showed a significant (p=0.032) loss of dopaminergic neurons. In spite of significant loss of dopaminergic neurons, DOPAL injections did not induce significant glial reaction in the substantia nigra. CONCLUSIONS: The present study provides the first in vivo quantification of substantia nigra pars compacta neuronal loss after injection of the endogenous toxin DOPAL. The results demonstrate that injections of DOPAL selectively kills SN DA neurons, suggests loss of striatal DA terminals, spares non-dopaminergic neurons of the pars reticulata, and triggers a behavioral phenotype (rotational asymmetry) consistent with other PD animal models. This study supports the "catecholaldehyde hypothesis" as an important link for the etiology of sporadic PD

Mouse models of neurodegenerative disease: preclinical imaging and neurovascular component.

Neurodegenerative diseases represent great challenges for basic science and clinical medicine because of their prevalence, pathologies, lack of mechanism-based treatments, and impacts on individuals. Translational research might contribute to the study of neurodegenerative diseases. The mouse has become a key model for studying disease mechanisms that might recapitulate in part some aspects of the corresponding human diseases. Neurode- generative disorders are very complicated and multifacto- rial. This has to be taken in account when testing drugs. Most of the drugs screening in mice are very di cult to be interpretated and often useless. Mouse models could be condiderated a ‘pathway models’, rather than as models for the whole complicated construct that makes a human disease. Non-invasive in vivo imaging in mice has gained increasing interest in preclinical research in the last years thanks to the availability of high-resolution single-photon emission computed tomography (SPECT), positron emission tomography (PET), high eld Magnetic resonance, Optical Imaging scanners and of highly speci c contrast agents. Behavioral test are useful tool to characterize di erent ani- mal models of neurodegenerative pathology. Furthermore, many authors have observed vascular pathological features associated to the di erent neurodegenerative disorders. Aim of this review is to focus on the di erent existing animal models of neurodegenerative disorders, describe behavioral tests and preclinical imaging techniques used for diagnose and describe the vascular pathological features associated to these diseases