Generation and genetic repair of two human induced pluripotent cell lines from patients with Epidermolysis Bullosa simplex and dilated cardiomyopathy associated with a heterozygous mutation in the translation initiation codon of KLHL24

Fibroblasts from two patients carrying a heterozygous mutation in the translation initiation codon (c.2 T > G) of the kelch-like protein 24 (KLHL24) gene were used to generate human induced pluripotent stem cells (hiPSCs), using non-integrating Sendai virus to deliver reprogramming factors. CRISPR-Cas9 editing was used for genetic correction of the mutation in the patient-hiPSCs. The top-predicted off-target sites were not altered. Patient and isogenic hiPSCs showed typical morphology, expressed pluripotency-associated markers, had the capacity for in vitro differentiation into the three germ layers and displayed a normal karyotype. These isogenic pairs will enable in vitro modelling of KLHL24-associated heart and skin conditions.Therapeutic cell differentiatio

Integrin α3β1 in hair bulge stem cells modulates CCN2 expression and promotes skin tumorigenesis

Epidermal-specific deletion of integrin α3β1 almost completely prevents the formation of papillomas during 7,12-Dimethylbenz [a]anthracene/12-O-tetradecanoylphorbol-13-acetate (DMBA/TPA) two-stage skin carcinogenesis. This dramatic decrease in tumorigenesis was thought to be due to an egress and premature differentiation of α3β1-depleted hair bulge (HB) stem cells (SCs), previously considered to be the cancer cells-of-origin in the DMBA/ TPA model. Using a reporter mouse line with inducible deletion of α3β1 in HBs, we show that HB SCs remain confined to their niche regardless of the presence of α3β1 and are largely absent from skin tumors. However, tumor formation was significantly decreased in mice deficient for α3β1 in HB SCs. RNA sequencing of HB SCs isolated from short-term DMBA/TPA-treated skin showed α3β1-dependent expression of the matricellular protein connective tissue growth factor (CCN2), which was confirmed in vitro, where CCN2 promoted colony formation and 3D growth of transformed keratinocytes. Together, these findings show that HBs contribute to skin tumorigenesis in an α3β1-dependent manner and suggest a role of HB SCs in creating a permissive environment for tumor growth through the modulation of CCN2 secretion

Can plantations develop understory biological and physical attributes of naturally regenerated forests?

With an increasing proportion of natural forests being replaced by plantations, there is a need to determine their potential to fulfill ecological purposes other than wood production. This study evaluated the extent to which deciduous and coniferous plantations develop understory attributes comparable to those of naturally regenerated stands. A functional group approach was used to synthesise species responses in terms of their ecological traits. Multivariate analyses of ecological traits revealed 16 emergent groups that shared common traits associated with a similar life history strategy. Responses of these groups, understory structure, and understory environmental conditions to plantation types and stand stages were analyzed and compared to naturally regenerated stands. Clear associations of trait responses to stand developmental stages and plantation types emerged. Light-demanding and wind-dispersed species groups were associated with early-successional stages, while woody groups, ferns and ant-dispersed spring-flowering herbs were associated with late-successional stages. Analyses also revealed an indicator group associated with old naturally regenerated forest. The understory functional groups and environmental conditions of deciduous plantations converged toward those of old naturally regenerated forests. However, understory structure in deciduous plantations remained poorly developed and richness of the indicator group was low compared to unplanted stands. Conifer plantations, currently the most common plantation type in the northern hardwood biome, showed a completely different pathway of understory development. Modifications to current plantation management practices are proposed to help recreate or maintain natural understory biological and physical attributes

Increased TIMP-3 expression alters the cellular secretome through dual inhibition of the metalloprotease ADAM10 and ligand-binding of the LRP-1 receptor

The tissue inhibitor of metalloproteinases-3 (TIMP-3) is a major regulator of extracellular matrix turnover and protein shedding by inhibiting different classes of metalloproteinases, including disintegrin metalloproteinases (ADAMs). Tissue bioavailability of TIMP-3 is regulated by the endocytic receptor low-density-lipoprotein receptor-related protein-1 (LRP-1). TIMP-3 plays protective roles in disease. Thus, different approaches have been developed aiming to increase TIMP-3 bioavailability, yet overall effects of increased TIMP-3 in vivo have not been investigated. Herein, by using unbiased mass-spectrometry we demonstrate that TIMP-3-overexpression in HEK293 cells has a dual effect on shedding of transmembrane proteins and turnover of soluble proteins. Several membrane proteins showing reduced shedding are known as ADAM10 substrates, suggesting that exogenous TIMP-3 preferentially inhibits ADAM10 in HEK293 cells. Additionally identified shed membrane proteins may be novel ADAM10 substrate candidates. TIMP-3-overexpression also increased extracellular levels of several soluble proteins, including TIMP-1, MIF and SPARC. Levels of these proteins similarly increased upon LRP-1 inactivation, suggesting that TIMP-3 increases soluble protein levels by competing for their binding to LRP-1 and their subsequent internalization. In conclusion, our study reveals that increased levels of TIMP-3 induce substantial modifications in the cellular secretome and that TIMP-3-based therapies may potentially provoke undesired, dysregulated functions of ADAM10 and LRP-1

Appendix A. A table listing the species codes used in Figs. 3 and 5.

A table listing the species codes used in Figs. 3 and 5

Generation and genetic repair of two human induced pluripotent stem cell lines from patients with Epidermolysis Bullosa simplex associated with a heterozygous mutation in the translation initiation codon of KLHL24

International audienc

Generation and genetic repair of two human induced pluripotent cell lines from patients with Epidermolysis Bullosa simplex and dilated cardiomyopathy associated with a heterozygous mutation in the translation initiation codon of KLHL24

Nonviral systems, such as lipid nanoparticles, have emerged as reliable methods to enable nucleic acid intracellular delivery. The use of cationic lipids in various formulations of lipid nanoparticles enables the formation of complexes with nucleic acid cargo and facilitates their uptake by target cells. However, due to their small size and highly charged nature, these nanocarrier systems can interact in vivo with antigen-presenting cells (APCs), such as dendritic cells (DCs) and macrophages. As this might prove to be a safety concern for developing therapies based on lipid nanocarriers, we sought to understand how they could affect the physiology of APCs. In the present study, we investigate the cellular and metabolic response of primary macrophages or DCs exposed to the neutral or cationic variant of the same lipid nanoparticle formulation. We demonstrate that macrophages are the cells affected most significantly and that the cationic nanocarrier has a substantial impact on their physiology, depending on the positive surface charge. Our study provides a first model explaining the impact of charged lipid materials on immune cells and demonstrates that the primary adverse effects observed can be prevented by fine-tuning the load of nucleic acid cargo. Finally, we bring rationale to calibrate the nucleic acid load of cationic lipid nanocarriers depending on whether immunostimulation is desirable with the intended therapeutic application, for instance, gene delivery or messenger RNA vaccines

Generation and genetic repair of two human induced pluripotent cell lines from patients with Epidermolysis Bullosa simplex and dilated cardiomyopathy associated with a heterozygous mutation in the translation initiation codon of KLHL24

Nonviral systems, such as lipid nanoparticles, have emerged as reliable methods to enable nucleic acid intracellular delivery. The use of cationic lipids in various formulations of lipid nanoparticles enables the formation of complexes with nucleic acid cargo and facilitates their uptake by target cells. However, due to their small size and highly charged nature, these nanocarrier systems can interact in vivo with antigen-presenting cells (APCs), such as dendritic cells (DCs) and macrophages. As this might prove to be a safety concern for developing therapies based on lipid nanocarriers, we sought to understand how they could affect the physiology of APCs. In the present study, we investigate the cellular and metabolic response of primary macrophages or DCs exposed to the neutral or cationic variant of the same lipid nanoparticle formulation. We demonstrate that macrophages are the cells affected most significantly and that the cationic nanocarrier has a substantial impact on their physiology, depending on the positive surface charge. Our study provides a first model explaining the impact of charged lipid materials on immune cells and demonstrates that the primary adverse effects observed can be prevented by fine-tuning the load of nucleic acid cargo. Finally, we bring rationale to calibrate the nucleic acid load of cationic lipid nanocarriers depending on whether immunostimulation is desirable with the intended therapeutic application, for instance, gene delivery or messenger RNA vaccines

Strong population structure in a species manipulated by humans since the Neolithic: The European fallow deer (Dama dama dama)

Species that have been translocated and otherwise manipulated by humans may show patterns of population structure that reflect those interactions. At the same time, natural processes shape populations, including behavioural characteristics like dispersal potential and breeding system. In Europe, a key factor is the geography and history of climate change through the Pleistocene. During glacial maxima throughout that period, species in Europe with temperate distributions were forced south, becoming distributed among the isolated peninsulas represented by Anatolia, Italy and Iberia. Understanding modern patterns of diversity depends on understanding these historical population dynamics. Traditionally, European fallow deer (Dama dama dama) are thought to have been restricted to refugia in Anatolia and possibly Sicily and the Balkans. However, the distribution of this species was also greatly influenced by human-mediated translocations. We focus on fallow deer to better understand the relative influence of these natural and anthropogenic processes. We compared modern fallow deer putative populations across a broad geographic range using microsatellite and mitochondrial DNA loci. The results revealed highly insular populations, depauperate of genetic variation and significantly differentiated from each other. This is consistent with the expectations of drift acting on populations founded by small numbers of individuals, and reflects known founder populations in the north. However, there was also evidence for differentiation among (but not within) physically isolated regions in the south, including Iberia. In those regions we find evidence for a stronger influence from natural processes than may be expected for a species with such strong, known anthropogenic influence. © 2017 Macmillan Publishers Limited, part of Springer Nature. All rights reserved