Development and large-scale production of human milk fat analog by fermentation of microalgae.

Background: Human milk contains a complex mixture of triacylglycerols (TAG), making it challenging to recreate using common ingredients. Objective: The study aimed to develop an innovative fermentation technique to produce essential human milk TAG, effectively tackling a significant hurdle in infant nutrition. Method: An in-depth analysis of the literature has been conducted to identify the specific TAG to be targeted. We used a microalgal oil production platform and a two-step procedure to modify its fatty acid and TAG composition. The palmitic acid (16:0) content has been increased by classical strain improvement techniques, followed by a step involving the expression of a lysophosphatidic acid acyltransferase (LPAAT) sequence capable of esterifying 16:0 specifically at the internal position (sn-2 palmitate) of TAG. Once the strain was stabilized, the fermentation was scaled up in a 50-L reactor to yield several kilograms of biomass. Subsequently, the oil was extracted and refined using standard oil processing conditions. Liquid chromatography-mass spectrometry was employed to monitor the TAG profile and the region specificity of 16:0 at the internal position (sn-2 palmitate) of TAG. Results: The initial strain had a 16:0 level of 25% of total fatty acids, which was increased to 30% by classical strain improvement. Simultaneously, the oleic acid level decreased from 61% to 57% of total fatty acids. Upon expression of an exogenous LPAAT gene, the level of the 16:0 esterified in the internal position of the TAG (sn-2 palmitate) increased by a factor of 10, to reach 73% of total palmitic acid. Consequently, the concentration of oleic acid in the internal position decreased from 81% to 22% of total fatty acids, with TAG analysis confirming that the primary TAG species in the oil was 1,3-dioleoyl-2-palmitoyl-glycerol (OPO). The 50-L-scale fermentation trial confirmed the strain's ability to produce oil with a yield of >150 g of oil per liter of fermentation broth in a timeframe of 5 days, rendering the process scalable for larger-scale industrialization. Conclusion: We have demonstrated the feasibility of producing a suitable TAG composition that can be effectively integrated into the formulations of infant nutrition in combination with other fats and oils to meet the infant feeding requirements.

Rasip1 regulates vertebrate vascular endothelial junction stability through Epac1-Rap1 signaling.

Establishment and stabilization of endothelial tubes with patent lumens is vital during vertebrate development. Ras-interacting protein 1 (RASIP1) has been described as an essential regulator of de novo lumenogenesis through modulation of endothelial cell (EC) adhesion to the extracellular matrix (ECM). Here, we show that in mouse and zebrafish embryos, Rasip1-deficient vessels transition from an angioblast cord to a hollow tube, permit circulation of primitive erythrocytes, but ultimately collapse, leading to hemorrhage and embryonic lethality. Knockdown of RASIP1 does not alter EC-ECM adhesion, but causes cell-cell detachment and increases permeability of EC monolayers in vitro. We also found that endogenous RASIP1 in ECs binds Ras-related protein 1 (RAP1), but not Ras homolog gene family member A or cell division control protein 42 homolog. Using an exchange protein directly activated by cyclic adenosine monophosphate 1 (EPAC1)-RAP1-dependent model of nascent junction formation, we demonstrate that a fraction of the RASIP1 protein pool localizes to cell-cell contacts. Loss of RASIP1 phenocopies loss of RAP1 or EPAC1 in ECs by altering junctional actin organization, localization of the actin-bundling protein nonmuscle myosin heavy chain IIB, and junction remodeling. Our data show that RASIP1 regulates the integrity of newly formed blood vessels as an effector of EPAC1-RAP1 signaling.

A mouse knockout library for secreted and transmembrane proteins.

Large collections of knockout organisms facilitate the elucidation of gene functions. Here we used retroviral insertion or homologous recombination to disrupt 472 genes encoding secreted and membrane proteins in mice, providing a resource for studying a large fraction of this important class of drug target. The knockout mice were subjected to a systematic phenotypic screen designed to uncover alterations in embryonic development, metabolism, the immune system, the nervous system and the cardiovascular system. The majority of knockout lines exhibited altered phenotypes in at least one of these therapeutic areas. To our knowledge, a comprehensive phenotypic assessment of a large number of mouse mutants generated by a gene-specific approach has not been described previously.

Kinome siRNA screen identifies regulators of ciliogenesis and hedgehog signal transduction.

Disruption or improper activation of the Hedgehog (Hh) pathway is associated with developmental abnormalities and cancer. Although characterized in Drosophila, the mechanisms that mediate the Hh signal downstream of the Smoothened (Smo) seven-transmembrane protein in vertebrates remain poorly understood. In particular, the Fused (Fu) kinase, which mediates Hh signaling in flies, is dispensable in mammals. To identify kinases that positively regulate the Hh pathway in mammals, we screened a mouse kinome small interfering RNA library and validated nine candidates that modulate Hh signaling. Among these candidates, Nek1 and Prkra did not directly function in the Hh pathway but exerted their effects on Hh signaling indirectly through a primary role in ciliogenesis. In contrast, another kinase, Cdc2l1, directly participated in the Hh pathway. Cdc2l1 was necessary and sufficient for activation of the Hh pathway, functioning downstream of Smo and upstream of the Glioma-associated (Gli) transcription factors. More specifically, Cdc2l1 interacted with the negative regulator Suppressor of Fused (Sufu) and relieved its inhibition on Gli, thus providing a mechanism for how Cdc2l1 might play a role in Hh signaling. Finally, with zebrafish as model organism, we showed that Cdc2l1 activated the Hh pathway in vivo. We propose that Cdc2l1 is a previously unrecognized member of the Hh signal transduction cascade.

Egfl7 knockdown causes defects in the extension and junctional arrangements of endothelial cells during zebrafish vasculogenesis.

The endothelial cell (EC) -specific secreted protein EGFL7 is important for tubulogenesis in newly forming blood vessels. We studied its role in vascular tube formation by a quantitative ultrastructural analysis of Egfl7-knockdown zebrafish embryos. At 24 hours postfertilization, the endothelia of dorsal aorta (DA) and posterior cardinal vein (PCV) were correctly anchored to the hypochord and endoderm, respectively, but failed to expand into the vascular area. This resulted in vessels with reduced or split lumen and open sheets of ECs. Concomitantly, the organization of hematopoietic cells-identified by the presence of previously undescribed membrane tubules-between DA and PCV, and within the vessels, was severely disturbed. Strikingly, ectopic cell junctions occurred across the obstructed vessel lumen, on the luminal EC surfaces, which in control conditions never display junctions of any kind. These data suggest that Egfl7 provides ECs with a cue for their extension into the vascular area and in establishing EC cell polarity.

The role of Egfl7 in vascular morphogenesis.

EGFL7 was identified by a number of groups as a putative secreted factor produced by the vascular endothelial cells (ECs). In a recent publication, we showed that EGFL7 regulates midline angioblast migration in zebrafish embryos-a key step in vascular tubulogenesis. In this study, we further characterized the zebrafish vasculature in the Egfl7 knockdown embryos at the ultrastructural level, and found that malformation of axial vessels is indeed due to the accumulation of angioblasts and aberrant connection among themselves, but not abnormal interaction between ECs and other cell types. Using in vitro biochemical assays, we demonstrated that EGFL7 is tightly associated with the extracellular matrix (ECM), and it supports EC migration either as a single factor or in combination with other ECM molecules. In order to evaluate if the biological function of EGFL7 is evolutionarily conserved, we generated Egfl7 knockout mice and analysed vascular development in a number of tissues. We found that vascular coverage of a given tissue is reduced or delayed, and vascular morphogenesis is defective in the Egfl7 mutant mice. Taken together, we conclude that EGFL7 provides a proper microenvironment for endothelial cell migration, thereby enabling accurate patterning. Our study indicates that the molecular composition of the ECM influences vascular morphogenesis.

The endothelial-cell-derived secreted factor Egfl7 regulates vascular tube formation.

Vascular development is a complex but orderly process that is tightly regulated. A number of secreted factors produced by surrounding cells regulate endothelial cell (EC) differentiation, proliferation, migration and coalescence into cord-like structures. Vascular cords then undergo tubulogenesis to form vessels with a central lumen. But little is known about how tubulogenesis is regulated in vivo. Here we report the identification and characterization of a new EC-derived secreted factor, EGF-like domain 7 (Egfl7). Egfl7 is expressed at high levels in the vasculature associated with tissue proliferation, and is downregulated in most of the mature vessels in normal adult tissues. Loss of Egfl7 function in zebrafish embryos specifically blocks vascular tubulogenesis. We uncover a dynamic process during which gradual separation and proper spatial arrangement of the angioblasts allow subsequent assembly of vascular tubes. This process fails to take place in Egfl7 knockdown embryos, leading to the failure of vascular tube formation. Our study defines a regulator that controls a specific and important step in vasculogenesis.