The loss of signalling between EphrinB2 and EphB4 in lymph vessels reflects the increase in vessel leakage seen in response to bacterial infections and in some genetic conditions such as lymphoedema.
Finding ways to control the signalling between these two proteins could help treat these conditions by developing drugs that improve endothelial cell integrity in lymph vessels. The barrier function of endothelial cells ECs varies among different organs and vessel types. The blood brain barrier BBB , for example, is formed of a continuous layer of ECs connected by specialized tight junctions and adherens junctions Zhao et al.
In contrast, blood vessels of the kidney and small intestine are lined by fenestrated ECs to facilitate rapid exchange, uptake and secretion of fluids, solutes and molecules Aird, The architecture of lymphatic endothelial cell LEC junctions also differs between the different vessel types Baluk et al.
Highly permeable button-like junctions of lymphatic capillaries allow uptake of fluid from the interstitium. The lymph is then drained and transported via collecting lymphatic vessels that are equipped with continuous zipper-like junctions preventing excessive leakage Baluk et al.
The molecular mechanisms that establish and maintain such functionally specialised junctional features of different vessel types are poorly understood.
Vascular integrity is regulated by junctional adhesion molecules, of which the adherens junction molecule vascular endothelial VE -cadherin and the tight junction molecule Claudin 5 CLDN5 have received particular attention. Dysregulation of VE-cadherin can result in junctional disruption, increased vessel permeability and severe lymph edema formation Frye et al. Interestingly, different effects of gene disruption in mice have been observed depending on the organ and the vessel type.
For example, endothelial loss of VE-cadherin in adult mice results in junctional disruption and increased vascular permeability in the heart and lung, but not the skin and brain Frye et al.
The junctional adhesion molecules are intracellularly associated to the actin cytoskeleton Dejana et al. The molecular control of endothelial cytoskeletal dynamics presents therefore a second important leverage point that regulates vascular integrity. Quiescent endothelia are characterized by a balance of actin stabilization and myosin-based actin pulling forces that are constantly applied to endothelial junctions.
Elevation of myosin-based actin contractility activates endothelial junctions. Differential expression and function of Rho GTPase regulators, such as the guanosine nucleotide exchange factor Vav3, has been proposed to contribute to barrier diversity across different blood vessels Hilfenhaus et al.
Upstream regulators of Rho GTPases may provide another level for organ- and vessel-type specific barrier regulation. Inducible EC-specific deletion of Efnb2 or Ephb4 disrupted lymphatic endothelial junctions in several organs while dermal and pulmonary blood vessel barrier function was not compromised. EphrinB2 and EphB4 play important roles in the embryonicand early postnatal development of blood and lymphatic vessels Adams et al.
We studied the effect on the dermal vasculature of the ear where Pdgfb-CreER T2 targets the endothelium of all blood vessels and collecting lymphatic vessels, but not lymphatic capillaries Wang et al.
A Experimental scheme for Ephb4 deletion in the mature vasculature by three consecutive intraperitoneal i. Mutant collecting vessels show abnormal LYVE1 expression. B Ear skin whole-mount immunofluorescence of 7-week-old Ephb4 mice using an antibody against VE-cadherin.
Note altered morphology of collecting lymphatic vessel junctions arrow in Ephb4 mutant compared to heterozygous littermates. D Ear skin whole-mount immunofluorescence of 5-week-old Efnb2 mice using an antibody against VE-cadherin. Note altered morphology of collecting lymphatic vessel junctions arrow in Efnb2 mutant compared to Cre negative littermates already after 2 weeks of Cre induction. E In vivo basal permeability assay in skin and lung of 5-week-old Efnb2 mutants and Cre negative littermates.
Efnb2 deletion does not impact on basal barrier function of skin and lung blood vasculature. Vascular leakage in the skin of 5-week-old Efnb2 mutants and Cre negative littermates was induced with VEGF or histamine. Note, endothelial deletion of Efnb2 does not impact on junctional regulation in leakage-induced dermal blood vasculature. VE-cadherin was used as a loading control.
Source data for panels E,F are provided. Unexpectedly, the cell-cell junctions of lymphatic capillaries not targeted by the Pdgfb-CreER T2 transgene also appeared disorganized in the Efnb2 mutant mice Figure 1—figure supplement 1B , suggesting secondary effects caused by disruption of collecting vessels.
Gross morphology of the blood vessels and the architecture of blood endothelial junctions were however unaltered in both mutants Figure 1A—D. We next performed a modified Miles assay, to assess whether barrier function of the blood endothelium was affected by endothelial deletion of Efnb2. In agreement with a lack of morphological alterations of the junctions, permeability of the skin or lung vasculature was not changed in Efnb2 mutants compared to control littermates under homeostasis Figure 1E.
Efficient depletion of EphrinB2 was shown exemplarily by Western blot from total lung lysates 8 days after the first tamoxifen administration Figure 1F. Gene deletion was induced by 4-OHT administration at postnatal day P 12, when remodeling of the ear vasculature into lymphatic capillaries and collecting vessels is initiated Lutter et al.
GFP reporter expression demonstrated efficient and specific Cre-mediated targeting of the lymphatic vasculature Figure 2A. Whole-mount staining for CLDN5 revealed disrupted cell-cell junctions and disintegration of the endothelial layer in Efnb2 -deficient collecting lymphatic vessels Figure 2A. Ultrastructural analysis of the Efnb2 mutant ear vasculature using transmission electron microscopy confirmed disruption of cell-cell junctions, characterized by large intercellular gaps Figure 2B,C and convoluted junctions with increased junction overlap compared to controls Figure 2B,D.
We also observed signs of cell degeneration Figure 2B. Deletion of Efnb2 in the quiescent dermal lymphatic vasculature at 8 weeks and analysis at 18 weeks of age also showed junctional alterations, albeit less severe than in the juvenile mice Figure 2—figure supplement 1B. GFP expression demonstrates Cre recombination in lymphatic endothelia. Note disintegration of collecting lymphatic vessels in Efnb2 and Ephb4 mutant mice arrows. B Ultrastructural analysis of the ear vasculature using transmission electron microscopy showing abnormal features of LEC junctions in Efnb2 mutant mice compared to heterozygous littermate: intercellular gaps asterisks , cell degeneration arrow and increased junction overlap white dotted line.
C,D Quantification of intercellular gaps and junctional overlap. Source data for panels C,D are provided. Analysis of the outer lymphatic endothelial layer of the subcapsular sinus of inguinal lymph nodes that forms during embryonic development Bovay et al.
In addition, analysis of the mesenteric vasculature of P11 Efnb2 and Ephb4 mice neonatally P4 treated with 4-OHT showed disorganisation of lymphatic endothelial cell-cell junctions in the mutant vessels compared to Efnb2 heterozygous or Cre-negative controls Figure 2F.
Analysis of the P11 neonatal mesenteric vasculature of the Efnb2 GFP reporter mice showed that Efnb2 expression was not restricted to valves but was present in all LECs of collecting vessels Figure 3A. In addition, defects in cell-cell junctions were observed in Efnb2 -deficient lymphatic vessel regions both upstream and downstream of the valve Figure 3B.
To quantify the state of the LEC junctions, we described four junctional categories based on previous definition of blood endothelial junctions Neto et al. Linear morphology was previously described to represent mature stable junctions while reticular and jagged morphologies indicate active remodeling Dorland and Huveneers, Finger-like structures were found in all lymphatic junctional categories, and were thus not included in the definition.
In wild type vessels, linear junctions were most frequent in both the upstream and downstream regions of the valve Figure 3C. Discontinuous junctions were not observed Figure 3C , and were thus considered pathological. Efnb2 -deficient vessels showed an almost complete loss of linear junctions at the expense of an increase in discontinuous junctions in collecting vessels, both upstream and downstream of the defective valves Figure 3C.
Source data for panels C,E are provided. To directly test if valve dysfunction caused by loss of Efnb2 contributes to the disruption of LEC junctions in collecting vessels, we generated a tamoxifen-inducible Cre line that specifically targets lymphatic valves. These results show that the disruption of lymphatic endothelial cell-cell junctions in Efnb2- deficient collecting vessels is not caused secondarily by valve dysfunction.
Single channel images are depicted in grey. There are many ways to enjoy this delicious beef korma. You can keep it completely simple and serve the beef korma with rice. Whenever I cook rice for an Indian meal, I like to put a couple of cardamom pods in the pot along with the rice, for that extra bit of exotic flavour. Instead of rice, you can also serve the beef korma with a nice naan bread. To get the full experience you could have a go at making your own naan. I have previously done it with my peshwari naan and the outcome was amazing.
If you are preparing a big dinner or a real Indian feast I highly recommend it. As always, the devil is in the detail. To give the beef korma the final touch, sprinkle a bit of fresh coriander on top of the plate when serving. This week one lucky reader will win one AnySharp Twist, sharpens any knife with diamond precision. With a fresh, modern look, just like its name you can certainly admire the curved silhouette of this latest model. Did you know?
The optimum angle for sharpening a good quality steel knife is 20 degrees? Which is why the Twist is preset to the same angle to allow for an optimum edge every time, leaving you with a professional finish in minutes. Replicate this easy beef korma recipe. Traditionally, a korma recipe is rich and creamy. With the addition of beef, it makes the korma all the more intense. Keywords: beef korma, Indian korma recipe, korma with coconut milk. Vegetable korma, it can be made differently each time depending on what vegetables are available.
This looks delicious! How many batches of the curry sauce base are needed to achieve 2 cups to use in this recipe? Hi Bec, My curry base sauce has 4 servings. I would make 2 batches of curry base sauce for 2 cups.
You might need extra as the sauce simmers. Best, Michelle. Sorry I found this very bland.. Then, the enriched libraries were eluted from the beads and were ready for a second cycle of hybridization. This hybridization step was necessary to obtain a wide specificity of regions of capture. The cluster density validation had been executed by the software of the instrument throughout the run.
Data obtained by next-generation sequencing NGS analysis were processed. Afterwards, the implant site was prepared with iodopovinone Betadine after trichotomy. Following a median sagittal incision of about 2. The skin flap was then sutured with Caprosyn synthetic monofilament adsorbable sutures Covidien AG, Neuhausen am Rheinfall, Switzerland using interrupted points.
Standard feeding and hydration were maintained as a constant throughout the postoperative phase. The investigation was carried out by means of a bright-field light microscope Leica Microsystem connected to a high-resolution digital camera DFCB Leica Leica Microsystem.
Histomorphometry, shown as the percentage of the newly formed bone, was carried out using a digitizing pad Matrix Vision GmbH, Oppenweiler, Germany and a histometry software package with image capturing capabilities Image-Pro Plus 4. Three-dimensional reconstruction was obtained by means of ZEN2 software Zeiss. CT was used to evaluate the bone repair.
The thickness of acquisition was 0. The 3D design of the five different generated scaffolds are shown in Fig. Surface morphology, pore and trabecular dimensions, and total porosity of the 3D printed scaffolds were characterized using SEM Fig. During printing, the scaffolds exhibited a reduction in real pore size and trabecular separation compared to their design, resulting in a concurrent reduction in total porosity.
There was also a reduction compared to planned values for design D and E Fig. Specific bands of RUNX2 were present in all samples of different design, while a higher expression of osteogenic-related marker is clearly visible in hGMSCs cultured with design scaffold C Fig. Microcomputed tomography three-dimensional rendering and evaluation of porosity, pore size, and wall thickness at different degradation time point.
Degradation in ascorbic acid solution was measured at day by the following changes in mass and pH. The lowest values in pH were recorded at day for all groups Fig. The various scaffold designs generally maintained their compressive mechanical properties during degradation in vitro.
On the other hand, design A preserved a significantly higher strength at the end of degradation compared with designs B and C at the same time point. Designs A and C showed the best performances in terms of strength after degradation Fig. In terms of yield strength at T 0 , design D showed significantly higher values compared with designs B, C, and E; specimens from design E had significantly lower values compared with the other four scaffolds.
On the other hand, design B showed a decrease in terms of yield strength Fig. As far as the compressive modulus at T 0 is concerned, design D showed the highest values, with a statistically significant difference compared with design C.
Design B showed a significant decrease over time. The metabolic activity of cells exposed to the extract of degrading PLA scaffolds was statistically different from the control group for all PBS extract concentrations, scaffold design, and degradation times.
Cells spread and extended on the uneven surface and across the filaments to create extended contact areas between them, organizing a multilayer covering the 3D substrate.
The scaffold material did not induce visible changes in cellular morphology. Although these size increases are particularly high for a simple coating of PEI, they also show the occurrence of a PEI coating. Figure 4 a1 highlights the presence of a large number of globular EVs of different dimensions with a central depression, thus confirming previous reports on the shape of EVs [ 39 ] as well as DLS data.
Some debris or aggregated vesicles were also observed. Very interestingly, the surface of EVs appeared relatively smooth.
At high magnification, the vesicles in the cytoplasmic compartment are clearly visible Fig. Low-magnification photographs were used to verify the Alizarin Red S staining Fig. In vitro osteogenic induction. EVs, extracellular vesicles; PEI, polyethyleneimine. Statistical analysis revealed that 31 genes were differentially expressed between the examined groups.
Finally, the comparison between the two big GO families ossification and osteoblast showed 19 genes that were common to the both families. Histological samples after staining with acid fuchsin and methylene blue solution showed a different response to various substrates. Histological evaluation. Acid fuchsin-toluidine blue staining. Arrows indicated bone defects. Histomorphometry analysis showed that newly formed bone represented Confocal laser scanning microscopy analysis.
Human GMSCs nuclei labeled with human anti-ANA green fluorescent conjugate are clearly visible in the rat calvaria, confirming the presence of living transplanted cells Fig. Right panels: Merged images of the two abovementioned channels.
Three-dimensional printing now allows fabrication of complex scaffold designs with different interconnections, porosity, and pore shape that were previously difficult to build [ 47 , 48 ]. The broad range of tested porosities represents the novelty of this work. These scaffolds were then doped with hGMSCs-derived extracellular vesicles and tested for their ability to regenerate bone defects induced in rat calvaria. In addition, absorbable polymers for bone tissue engineering must ensure mechanical stability while degrading, thus keeping defect site stability during bone regeneration [ 49 , 50 ].
We examined here which 3D-PLA printed porous scaffold designs provided the best mechanical stability over time during their degradation.
SEM examination revealed that the 3D PLA printed scaffolds showed larger filaments and reduced pore size with respect to the features of the PLA polymer during extrusion and subsequent cooling. As a consequence, the scaffolds produced in this study should show adequate cellular migration and provide nutrients after in vivo implantation [ 53 ].
Since the influence of nanoscale topography is crucial for bone substitute biomaterials, its role on our 3D-PLA scaffolds must be considered [ 54 ].
The scaffold degradation was revealed by mass loss and decrease of extract pH, and tested at increasing times over a days period. At the end of the study, the mass was significantly reduced in all groups.
The pH reduction confirmed the lactide loss [ 55 ]. Noteworthy, the degradation did not have a major impact on the mechanical properties over the days time frame. Scaffold-host tissue compatibility is a fundamental characteristic. Therefore, when using a degradable material its degradation byproducts must not induce a cytotoxic response. This work evaluated the potential cytotoxicity elicited during the absorption of PLA.
Lactic acid is one of the PLA hydrolytical degradation byproducts [ 56 ]. This is a key point, as one indication of degradation of polyesters is a decrease in pH as seen in this study. The reduced cell metabolic activity after incubation with the eluates was statistically lower than control cells; however, it must be considered that this result was not influenced by the increasing degradation time for scaffold design C. The slow absorption of the tested PLA allowed for mechanical stability over the estimated bone formation and maturation time.
Moreover, this pattern reduced the cytotoxicity potential due to slow release of acid. The slow absorption rate and host metabolization of lactic acid are fundamental for a low cytotoxic response in vivo. For PLA in vivo, the acidic byproduct is lactic acid and it would be metabolized through the Cori cycle; this process cannot be replicated in vitro [ 57 ]. Among the various tissues from which MSCs can be isolated, growing attention has been paid to dental tissues including periodontal ligament, dental pulp, and gingiva owing to the minimally invasive procedure involved in their collection, and remarkable differentiation ability towards neurogenic and other cells [ 60 ].
Recently, animal data confirm that periodontal ligament stem cells seeded onto 3D scaffolds in the mouse calvaria undergo precocious osteointegration and vascularization [ 36 , 61 ]. This effect may be due to the ability of the stem cells to undergo osteogenic differentiation, but also to their immunomodulatory and anti-inflammatory properties.
Released membrane vesicles from eukaryotic cells, as exosomes, microparticles, microvesicles, and apoptotic bodies, can be retained as a dynamic extracellular vesicular compartment, strategic for their paracrine or autocrine biological effects in tissue metabolism [ 62 ]. In vitro, mineralizing osteoblast exosomes are capable of entering into bone marrow stromal cells to induce them to the osteoblast phenotype, with trough upregulation of b-catenin in recipient cells representing a potential therapeutic approach [ 64 ].
As far as PEI-engineered EVs are concerned, many factors such as molecular weight, degree of branching, zeta potential, particle size, cationic charge density, molecular structure, sequence, and conformational flexibility have all been shown to affect PEI efficiency and cytotoxicity when used as a transfecting agent [ 66 ].
We found that the best compromise between effectiveness and toxicity was a concentration of 0. The subsequent internalization mechanisms proposed for EVs are attachment or fusion with the target cell membrane, delivering exosomal proteins to the recipient cell [ 68 , 69 ], or internalization by the recipient cells by mechanisms such as endocytosis [ 70 ].
In the present study, labeled PEI-EVs were demonstrated to be internalized mainly by endocytosis, being highly represented after incubation in the recipient cells. In fact, mounting evidence suggests that EVs alone are capable of promoting proliferation and migration of cells as well as osteogenesis and angiogenesis [ 72 ]. Our results showed that 31 genes were differentially expressed between the examined groups. In light of the above findings, we investigated the osteogenic effects of 3D-PLA scaffold infiltrated or not with hGMSCs-derived extracellular vesicles.
In vivo studies testing biomaterials in the complex environment of the body are considered strategic to understanding the biological machinery involved in bone regeneration. In addition, the presence of the EVs and, more specifically, PEI-EVs linked to the 3D-PLA scaffold improve the mineralization process as well as the development of an extensive vascular network that is indicative of osteointegration, as recently shown by Xie et al.
Precocious puberty, prostatic carcinoma or other androgen-dependent neoplasm, prior allergic reaction to HCG. HCG should be used in conjunction with human menopausal gonadotropins only by physicians experienced with infertility problems who are familiar with the criteria for patient selection, contraindications, warnings, precautions, and adverse reactions described in the package insert for menotropins.
Since androgens may cause fluid retention, HCG should be used with caution in patients with cardiac or renal disease, epilepsy, migraine, or asthma.
Induction of androgen secretion by HCG may induce precocious puberty in pediatric patients treated for cryptorchidism. Therapy should be discontinued if signs of precocious puberty occur.
Headache, irritability, restlessness, depression, fatigue, edema, precocious puberty, gynecomastia, pain at the site of injection. For intramuscular use only. The dosage regimen employed in any particular case will depend upon the indication for the use, the age and weight of the patient, and the physician's preference.
The following regimens have been advocated by various authorities:. Prepubertal cryptorchidism not due to anatomical obstruction. Therapy is usually instituted in children between the ages of 4 and 9. Selected cases of hypogonadotropic hypogonadism in males. Induction of ovulation and pregnancy in the anovulatory, infertile woman in whom the cause of anovulation is secondary and not due to primary ovarian failure and who has been appropriately pretreated with human menotropins.
See prescribing information for menotropins for dosage and administration for that drug product. A dosage of 10, USP units is recommended in the labeling for menotropins.MERCK MIX3: Various: Pain In My Gulliver Various - Merck Mix 3 (CD, Mixed) Merck: MERCK MIX3: US: Sell This Version: 1 – 4 of 4. Show. Reviews Add Review. Lists Add to .