Newly developed genome-editing tools, such as the clustered regularly interspaced short palindromic repeat (CRISPR)-Cas9 system, allow simple and rapid genetic modification in most model organisms ...and human cell lines. Here, we report the production and analysis of mice carrying the inactivation via deletion of a genomic insulator, a key non-coding regulatory DNA element found 5' upstream of the mouse tyrosinase (Tyr) gene. Targeting sequences flanking this boundary in mouse fertilized eggs resulted in the efficient deletion or inversion of large intervening DNA fragments delineated by the RNA guides. The resulting genome-edited mice showed a dramatic decrease in Tyr gene expression as inferred from the evident decrease of coat pigmentation, thus supporting the functionality of this boundary sequence in vivo, at the endogenous locus. Several potential off-targets bearing sequence similarity with each of the two RNA guides used were analyzed and found to be largely intact. This study reports how non-coding DNA elements, even if located in repeat-rich genomic sequences, can be efficiently and functionally evaluated in vivo and, furthermore, it illustrates how the regulatory elements described by the ENCODE and EPIGENOME projects, in the mouse and human genomes, can be systematically validated.
Cell biologists generally consider that microtubules and actin play complementary roles in long- and short-distance transport in animal cells. On the contrary, using melanosomes of melanocytes as a ...model, we recently discovered that the motor protein myosin-Va works with dynamic actin tracks to drive long-range organelle dispersion in opposition to microtubules. This suggests that in animals, as in yeast and plants, myosin/actin can drive long-range transport. Here, we show that the SPIRE-type actin nucleators (predominantly SPIRE1) are Rab27a effectors that co-operate with formin-1 to generate actin tracks required for myosin-Va-dependent transport in melanocytes. Thus, in addition to melanophilin/myosin-Va, Rab27a can recruit SPIREs to melanosomes, thereby integrating motor and track assembly activity at the organelle membrane. Based on this, we suggest a model in which organelles and force generators (motors and track assemblers) are linked, forming an organelle-based, cell-wide network that allows their collective activity to rapidly disperse the population of organelles long-distance throughout the cytoplasm.
Although calcineurin inhibitors are very effective as immunosuppressants in organ transplantation, complete graft acceptance remains as a challenge. Transfer of genes with immunosuppressant functions ...could contribute to improving the clinical evolution of transplantation. In this sense, hydrodynamic injection has proven very efficacious for liver gene transfer. In the present work, the
gene was hydrofected 'ex vivo' to pig livers during the bench surgery stage, to circumvent the cardiovascular limitations of the procedure, in a model of porcine orthotopic transplantation with a 10-day follow-up. We used IL-10 because human and porcine proteins can be differentially quantified and for its immunomodulatory pleiotropic functions. Safety (biochemical parameters and histology), expression efficacy (RNA transcription and blood protein expression), and acute inflammatory response (cytokines panel) of the procedure were evaluated. The procedure proved safe as no change in biochemical parameters was observed in treated animals, and human IL-10 was efficaciously expressed, with stationary plasma protein levels over 20 pg/mL during the follow-up. Most studied cytokines showed increments (interferon-α, IFN-α; interleukin-1β, IL-1β; tumor necrosis factor α, TNFα; interleukin-6, IL-6; interleukin-8, IL-8; interleukin-4, IL-4; and transforming growth factor-β, TGF-β) in treated animals, without deleterious effects on tissue. Collectively, the results support the potential clinical interest in this gene therapy model that would require further longer-term dose-response studies to be confirmed.
Inherited hearing impairment is a remarkably heterogeneous monogenic condition, involving hundreds of genes, most of them with very small (< 1%) epidemiological contributions. The exception is GJB2, ...the gene encoding connexin-26 and underlying DFNB1, which is the most frequent type of autosomal recessive non-syndromic hearing impairment (ARNSHI) in most populations (up to 40% of ARNSHI cases). DFNB1 is caused by different types of pathogenic variants in GJB2, but also by large deletions that keep the gene intact but remove an upstream regulatory element that is essential for its expression. Such large deletions, found in most populations, behave as complete loss-of-function variants, usually associated with a profound hearing impairment. By using CRISPR-Cas9 genetic edition, we have generated a murine model (Dfnb1
) that reproduces the most frequent of those deletions, del(GJB6-D13S1830). Dfnb1
homozygous mice are viable, bypassing the embryonic lethality of the Gjb2 knockout, and present a phenotype of profound hearing loss (> 90 dB SPL) that correlates with specific structural abnormalities in the cochlea. We show that Gjb2 expression is nearly abolished and its protein product, Cx26, is nearly absent all throughout the cochlea, unlike previous conditional knockouts in which Gjb2 ablation was not obtained in all cell types. The Dfnb1
model recapitulates the clinical presentation of patients harbouring the del(GJB6-D13S1830) variant and thus it is a valuable tool to study the pathological mechanisms of DFNB1 and to assay therapies for this most frequent type of human ARNSHI.
Abstract This article examines artificial intelligence (AI) co-regulation in the EU AI Act and the critical role of standards under this regulatory strategy. It engages with the foundation of ...democratic legitimacy in EU standardization, emphasizing the need for reform to keep pace with the rapid evolution of AI capabilities, as recently suggested by the European Parliament. The article highlights the challenges posed by interdisciplinarity and the lack of civil society expertise in standard-setting. It critiques the inadequate representation of societal stakeholders in the development of AI standards, posing pressing questions about the potential risks this entails to the protection of fundamental rights, given the lack of democratic oversight and the global composition of standard-developing organizations. The article scrutinizes how under the AI Act technical standards will define AI risks and mitigation measures and questions whether technical experts are adequately equipped to standardize thresholds of acceptable residual risks in different high-risk contexts. More specifically, the article examines the complexities of regulating AI, drawing attention to the multi-dimensional nature of identifying risks in AI systems and the value-laden nature of the task. It questions the potential creation of a typology of AI risks and highlights the need for a nuanced, inclusive, and context-specific approach to risk identification and mitigation. Consequently, in the article we underscore the imperative for continuous stakeholder involvement in developing, monitoring, and refining the technical rules and standards for high-risk AI applications. We also emphasize the need for rigorous training, certification, and surveillance measures to ensure the enforcement of fundamental rights in the face of AI developments. Finally, we recommend greater transparency and inclusivity in risk identification methodologies, urging for approaches that involve stakeholders and require a diverse skill set for risk assessment. At the same time, we also draw attention to the diversity within the European Union and the consequent need for localized risk assessments that consider national contexts, languages, institutions, and culture. In conclusion, the article argues that co-regulation under the AI Act necessitates a thorough re-examination and reform of standard-setting processes, to ensure a democratically legitimate, interdisciplinary, stakeholder-inclusive, and responsive approach to AI regulation, which can safeguard fundamental rights and anticipate, identify, and mitigate a broad spectrum of AI risks.
Rab GTPases are compartment-specific molecular switches that regulate intracellular vesicular transport in eukaryotes. GDP/GTP exchange factors (GEFs) control Rab activation, and current models ...propose that localised and regulated GEF activity is important in targeting Rabs to specific membranes. Here, we investigated the mechanism of GEF function using the Rab27a GEF, Rab3GEP (also known as MADD), in melanocytes as a model. We show that Rab3GEP-deficient melanocytes (melan-R3G
) manifest partial disruption of melanosome dispersion, a read-out of Rab27a activation and targeting. Using rescue of melanosome dispersion in melan-R3G
cells and effector pull-down approaches we show that the DENN domain of Rab3GEP (conserved among RabGEFs) is necessary, but insufficient, for its cellular function and GEF activity. Finally, using a mitochondrial re-targeting strategy, we show that Rab3GEP can target Rab27a to specific membranes in a GEF-dependent manner. We conclude that Rab3GEP facilitates the activation and targeting of Rab27a to specific membranes, but that it differs from other DENN-containing RabGEFs in requiring DENN and non-DENN elements for both of these activities and by lacking compartment-specific localisation.
In animal cells, microtubule and actin tracks and their associated motors (dynein, kinesin, and myosin) are thought to regulate long- and short-range transport, respectively 1–8. Consistent with ...this, microtubules extend from the perinuclear centrosome to the plasma membrane and allow bidirectional cargo transport over long distances (>1 μm). In contrast, actin often comprises a complex network of short randomly oriented filaments, suggesting that myosin motors move cargo short distances. These observations underpin the “highways and local roads” model for transport along microtubule and actin tracks 2. The “cooperative capture” model exemplifies this view and suggests that melanosome distribution in melanocyte dendrites is maintained by long-range transport on microtubules followed by actin/myosin-Va-dependent tethering 5, 9. In this study, we used cell normalization technology to quantitatively examine the contribution of microtubules and actin/myosin-Va to organelle distribution in melanocytes. Surprisingly, our results indicate that microtubules are essential for centripetal, but not centrifugal, transport. Instead, we find that microtubules retard a centrifugal transport process that is dependent on myosin-Va and a population of dynamic F-actin. Functional analysis of mutant proteins indicates that myosin-Va works as a transporter dispersing melanosomes along actin tracks whose +/barbed ends are oriented toward the plasma membrane. Overall, our data highlight the role of myosin-Va and actin in transport, and not tethering, and suggest a new model in which organelle distribution is determined by the balance between microtubule-dependent centripetal and myosin-Va/actin-dependent centrifugal transport. These observations appear to be consistent with evidence coming from other systems showing that actin/myosin networks can drive long-distance organelle transport and positioning 10, 11.
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•Microtubules are essential for centripetal, but not centrifugal, melanosome transport•Myosin-Va and a dynamic actin pool drive long-range centrifugal melanosome transport•Myosin-Va is a processive plus-end-directed motor, and not a tether, in melanocytes•Opposing myosin-Va/actin and microtubule forces regulate melanosome distribution
In animal cells, microtubules and actin are thought to regulate long- and short-range transport, respectively. Here, Evans et al. test the contribution of these systems to organelle transport using melanocyte pigment granules as a model. Surprisingly, they find that myosin-Va and dynamic actin drive long-range transport to the membrane.
Yb and Er codoped NaT(XO4)2 (T = Y, La, Gd, Lu and X = Mo, W) disordered oxides show a green (Er3+ related) up-conversion (UC) efficiency comparable to that of Yb:Er:β-NaYF4 compound and unless 3 ...times larger UC ratiometric thermal sensitivity. The similar UC efficiency of Yb:Er doped NaT(XO4)2 and β-NaYF4 compounds allowed testing equal subcutaneous depths of ex-vivo chicken tissue in both cases. This extraordinary behavior for NaT(XO4)2 oxides with large cutoff phonon energy (ħω≈ 920 cm-1) is ascribed to 4F9/2 electron population recycling to higher energy 4G11/2 level by a phonon assisted transition. Crystalline nanoparticles of Yb:Er:NaLu(MoO4)2 have been synthesized by sol-gel with sizes most commonly in the 50-80 nm range, showing a relatively small reduction of the UC efficiency with regards to bulk materials. Fluorescence lifetime and multiphoton imaging microscopies show that these nanoparticles can be efficiently distributed to all body organs of a perfused mouse.
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