MMP-12, Secreted by Pro-Inflammatory Macrophages, Targets Endoglin in Human Macrophages and Endothelial Cells

Upon inflammation, monocyte-derived macrophages (MF) infiltrate blood vessels to regulate several processes involved in vascular pathophysiology. However, little is known about the mediators involved. Macrophage polarization is crucial for a fast and e cient initial response (GM-MF) and a good resolution (M-MF) of the inflammatory process. The functional activity of polarized MF is exerted mainly through their secretome, which can target other cell types, including endothelial cells. Endoglin (CD105) is a cell surface receptor expressed by endothelial cells and MF that is markedly upregulated in inflammation and critically involved in angiogenesis. In addition, a soluble form of endoglin with anti-angiogenic activity has been described in inflammation-associated pathologies. The aim of this work was to identify components of the MF secretome involved in the shedding of soluble endoglin. We find that the GM-MF secretome contains metalloprotease 12 (MMP-12), a GM-MF specific marker that may account for the anti-angiogenic activity of the GM-MF secretome. Cell surface endoglin is present in both GM-MF and M-MF, but soluble endoglin is only detected in GM-MF culture supernatants. Moreover, MMP-12 is responsible for the shedding of soluble endoglin in vitro and in vivo by targeting membrane-bound endoglin in both MF and endothelial cells. These data demonstrate a direct correlation between GM-MF polarization, MMP-12, and soluble endoglin expression and function. By targeting endothelial cells, MMP-12 may represent a novel mediator involved in vascular homeostasis.Ministerio de Ciencia, Innovación y Universidades of Spain (SAF2013-43421-R to C.B.; SAF2017-83785-R and SAF2014-23801 to A.L.C.)Consejo Superior de Investigaciones Cientificas (201920E022 to C.B.)Centro de Investigación Biomédica en Red de Enfermedades Raras (CIBERER; ISCIII-CB06/07/0038 to C.B.)Czech Republic Specific University Research (SVV-260414 to P.N.)CIBERER is an initiative of the Instituto de Salud Carlos III (ISCIII) of Spain supported by FEDER fundsM.A. was funded with a fellowship from Ministerio de Ciencia e Innovación (BES-2008-003888)M.V. was supported by a short-term mobility fellowship from the European Erasmus Programm

Gene Editing in Adult Hematopoietic Stem Cells

Over the last years, an important development has allowed the scientific community to address a precise and accurate modification of the genome. The first probe of concept appeared with the design and use of engineered zinc-finger nucleases (ZFNs), which was expanded later on with the discovery and engineering of meganucleases and transcription activator-like effector nucleases (TALENs) and finally democratized and made easily available to the whole scientific community with the discovery of the clustered regularly interspaced short palindromic repeats (CRISPR)/Cas9 nuclease combination technology. The availability of these tools has allowed a precise gene editing, such as knockout of a specific gene or the correction of a defective gene by means of homologous recombination (HR), taking advantage of the endogenous cell repair machinery. This process was already known and used but was inefficient—efficiency that has been increased more than 100-fold with the addition of the mentioned specific nucleases to the process. Apart from the proper design of the nucleases to recognize and cut the selected site in the cell genome, two main goals need to be adequately addressed to optimize its function: the delivery of the tools into the desired cells and the selection of those where the gene editing process has occurred correctly. Both steps can be easily solved when the source of cells is extensive or can be expanded and manipulated in vitro extensively, such as immortalized cell lines or pluripotent stem cells (embryonic stem cells and induced pluripotent stem cells). However, both steps are critical in the case of primary cells, such as the hematopoietic stem cells (HSCs). HSCs are a rare cell population present in the bone marrow (BM) of higher mammals, and it is the responsible for the maintenance and replenishment of all hematopoietic cells for the lifespan of the animals by means of two fundamental properties: self-renewal and multipotency. HSC population is then the ideal target for the correction of hematopoietic genetic diseases and also for the knockout of the responsible genes to in vitro and in vivo model those hematopoietic diseases. This rare population cannot be expanded and its in vitro manipulation and culture negatively affects their fundamental properties of self-renewal and multipotency. These factors challenge the application of gene editing to HSCs. Important efforts are now ongoing trying to optimize the protocols of gene delivery and selection for HSCs. This chapter will review and discuss how researchers are trying to solve them, all attempts that are ongoing and the potential application of the technology to the patients affected with hematopoietic genetic diseases

Abiotic conditions in cephalopod (Sepia officinalis) eggs: embryonic development at low pH and high pCO2

Low pO(2) values have been measured in the perivitelline fluids (PVF) of marine animal eggs on several occasions, especially towards the end of development, when embryonic oxygen consumption is at its peak and the egg case acts as a massive barrier to diffusion. Several authors have therefore suggested that oxygen availability is the key factor leading to hatching. However, there have been no measurements of PVF pCO(2) so far. This is surprising, as elevated pCO(2) could also constitute a major abiotic stressor for the developing embryo. As a first attempt to fill this gap in knowledge, we measured pO(2), pCO(2) and pH in the PVF of late cephalopod (Sepia officinalis) eggs. We found linear relationships between embryo wet mass and pO(2), pCO(2) and pH. pO(2) declined from > 12 kPa to less than 5 kPa, while pCO(2) increased from 0.13 to 0.41 kPa. In the absence of active accumulation of bicarbonate in the PVF, pH decreased from 7.7 to 7.2. Our study supports the idea that oxygen becomes limiting in cephalopod eggs towards the end of development; however, pCO(2) and pH shift to levels that have caused significant physiological disturbances in other marine ectothermic animals. Future research needs to address the physiological adaptations that enable the embryo to cope with the adverse abiotic conditions in their egg environment

P5CS expression study in a new family with ALDH18A1-associated hereditary spastic paraplegia SPG9

In 2015\u20132016, we and others reported ALDH18A1 mutations causing dominant (SPG9A) or recessive (SPG9B) spastic paraplegia. In vitro production of the ALDH18A1 product, \u3941-pyrroline-5-carboxylate synthetase (P5CS), appeared necessary for cracking SPG9 disease-causing mechanisms. We now describe a baculovirus\u2013insect cell system that yields mgs of pure human P5CS and that has proven highly valuable with two novel P5CS mutations reported here in new SPG9B patients. We conclude that both mutations are disease-causing, that SPG9B associates with partial P5CS deficiency and that it is clinically more severe than SPG9A, as reflected in onset age, disability, cognitive status, growth, and dysmorphic traits

Transforming growth factor beta signaling: The master sculptor of fingers

Transforming growth factor beta (TGF?) constitutes a large and evolutionarily conserved superfamily of secreted factors that play essential roles in embryonic development, cancer, tissue regeneration, and human degenerative pathology. Studies of this signaling cascade in the regulation of cellular and tissue changes in the three-dimensional context of a developing embryo have notably advanced in the understanding of the action mechanism of these growth factors. In this review, we address the role of TGF? signaling in the developing limb, focusing on its essential function in the morphogenesis of the autopod. As we discuss in this work, modern mouse genetic experiments together with more classical embryological approaches in chick embryos, provided very valuable information concerning the role of TGF? and Activin family members in the morphogenesis of the digits of tetrapods, including the formation of phalanxes, digital tendons, and interphalangeal joints. We emphasize the importance of the Activin and TGF? proteins as digit inducing factors and their critical interaction with the BMP signaling to sculpt the hand and foot morphology

The Vinculin-ΔIn20/21 Mouse: Characteristics of a Constitutive, Actin-Binding Deficient Splice Variant of Vinculin

BACKGROUND: The cytoskeletal adaptor protein vinculin plays a fundamental role in cell contact regulation and affects central aspects of cell motility, which are essential to both embryonal development and tissue homeostasis. Functional regulation of this evolutionarily conserved and ubiquitously expressed protein is dominated by a high-affinity, autoinhibitory head-to-tail interaction that spatially restricts ligand interactions to cell adhesion sites and, furthermore, limits the residency time of vinculin at these sites. To date, no mutants of the vinculin protein have been characterized in animal models. METHODOLOGY/PRINCIPAL FINDINGS: Here, we investigate vinculin-DeltaEx20, a splice variant of the protein lacking the 68 amino acids encoded by exon 20 of the vinculin gene VCL. Vinculin-DeltaEx20 was found to be expressed alongside with wild type protein in a knock-in mouse model with a deletion of introns 20 and 21 (VCL-DeltaIn20/21 allele) and shows defective head-to-tail interaction. Homozygous VCL-DeltaIn20/21 embryos die around embryonal day E12.5 showing cranial neural tube defects and exencephaly. In mouse embryonic fibroblasts and upon ectopic expression, vinculin-DeltaEx20 reveals characteristics of constitutive head binding activity. Interestingly, the impact of vinculin-DeltaEx20 on cell contact induction and stabilization, a hallmark of the vinculin head domain, is only moderate, thus allowing invasion and motility of cells in three-dimensional collagen matrices. Lacking both F-actin interaction sites of the tail, the vinculin-DeltaEx20 variant unveils vinculin's dynamic binding to cell adhesions independent of a cytoskeletal association, and thus differs from head-to-tail binding deficient mutants such as vinculin-T12, in which activated F-actin binding locks the protein variant to cell contact sites. CONCLUSIONS/SIGNIFICANCE: Vinculin-DeltaEx20 is an active variant supporting adhesion site stabilization without an enhanced mechanical coupling. Its presence in a transgenic animal reveals the potential of splice variants in the vinculin gene to alter vinculin function in vivo. Correct control of vinculin is necessary for embryonic development

Solving patients with rare diseases through programmatic reanalysis of genome-phenome data

Reanalysis of inconclusive exome/genome sequencing data increases the diagnosis yield of patients with rare diseases. However, the cost and efforts required for reanalysis prevent its routine implementation in research and clinical environments. The Solve-RD project aims to reveal the molecular causes underlying undiagnosed rare diseases. One of the goals is to implement innovative approaches to reanalyse the exomes and genomes from thousands of well-studied undiagnosed cases. The raw genomic data is submitted to Solve-RD through the RD-Connect Genome-Phenome Analysis Platform (GPAP) together with standardised phenotypic and pedigree data. We have developed a programmatic workflow to reanalyse genome-phenome data. It uses the RD-Connect GPAP’s Application Programming Interface (API) and relies on the big-data technologies upon which the system is built. We have applied the workflow to prioritise rare known pathogenic variants from 4411 undiagnosed cases. The queries returned an average of 1.45 variants per case, which first were evaluated in bulk by a panel of disease experts and afterwards specifically by the submitter of each case. A total of 120 index cases (21.2% of prioritised cases, 2.7% of all exome/genome-negative samples) have already been solved, with others being under investigation. The implementation of solutions as the one described here provide the technical framework to enable periodic case-level data re-evaluation in clinical settings, as recommended by the American College of Medical Genetics

Evaluation of different bowel preparations for small bowel capsule endoscopy: a prospective, randomized, controlled study

To obtain an adequate view of the whole small intestine during capsule endoscopy (CE) a clear liquid diet and overnight fasting is recommended. However, intestinal content can hamper vision in spite of these measures. Our aim was to evaluate tolerance and degree of intestinal cleanliness during CE following three types of bowel preparation. PATIENTS AND METHODS: This was a prospective, multicenter, randomized, controlled study. Two-hundred ninety-one patients underwent one of the following preparations: 4 L of clear liquids (CL) (group A; 92 patients); 90 mL of aqueous sodium phosphate (group B; 89 patients); or 4 L of a polyethylene glycol electrolyte solution (group C; 92 patients). The degree of cleanliness of the small bowel was classified by blinded examiners according to four categories (excellent, good, fair or poor). The degree of patient satisfaction, gastric and small bowel transit times, and diagnostic yield were measured. RESULTS: The degree of cleanliness did not differ significantly between the groups (P = 0.496). Interobserver concordance was fair (k = 0.38). No significant differences were detected between the diagnostic yields of the CE (P = 0.601). Gastric transit time was 35.7 +/- 3.7 min (group A), 46.1 +/- 8.6 min (group B) and 34.6 +/- 5.0 min (group C) (P = 0.417). Small-intestinal transit time was 276.9 +/- 10.7 min (group A), 249.7 +/- 13.1 min (group B) and 245.6 +/- 11.6 min (group C) (P = 0.120). CL was the best tolerated preparation. Compliance with the bowel preparation regimen was lowest in group C (P = 0.008). CONCLUSIONS: A clear liquid diet and overnight fasting is sufficient to achieve an adequate level of cleanliness and is better tolerated by patients than other forms of preparation

Is telomere length socially patterned? Evidence from the West of Scotland Twenty-07 study

Lower socioeconomic status (SES) is strongly associated with an increased risk of morbidity and premature mortality, but it is not known if the same is true for telomere length, a marker often used to assess biological ageing. The West of Scotland Twenty-07 Study was used to investigate this and consists of three cohorts aged approximately 35 (N = 775), 55 (N = 866) and 75 years (N = 544) at the time of telomere length measurement. Four sets of measurements of SES were investigated: those collected contemporaneously with telomere length assessment, educational markers, SES in childhood and SES over the preceding twenty years. We found mixed evidence for an association between SES and telomere length. In 35-year-olds, many of the education and childhood SES measures were associated with telomere length, i.e. those in poorer circumstances had shorter telomeres, as was intergenerational social mobility, but not accumulated disadvantage. A crude estimate showed that, at the same chronological age, social renters, for example, were nine years (biologically) older than home owners. No consistent associations were apparent in those aged 55 or 75. There is evidence of an association between SES and telomere length, but only in younger adults and most strongly using education and childhood SES measures. These results may reflect that childhood is a sensitive period for telomere attrition. The cohort differences are possibly the result of survival bias suppressing the SES-telomere association; cohort effects with regard different experiences of SES; or telomere possibly being a less effective marker of biological ageing at older ages