In Vivo Evaluation of Cervical Stiffness Evolution during Induced Ripening Using Shear Wave Elastography,Histology and 2 Photon Excitation Microscopy: Insight from an Animal Model

Prematurity affects 11% of the births and is the main cause of infant mortality. On the opposite case, the failure of induction of parturition in the case of delayed spontaneous birth is associated with fetal suffering. Both conditions are associated with precocious and/or delayed cervical ripening. Quantitative and objective information about the temporal evolution of the cervical ripening may provide a complementary method to identify cases at risk of preterm delivery and to assess the likelihood of successful induction of labour. In this study, the cervical stiffness was measured in vivo in pregnant sheep by using Shear Wave Elastography (SWE). This technique assesses the stiffness of tissue through the measurement of shear waves speed (SWS). In the present study, 9 pregnant ewes were used. Cervical ripening was induced at 127 days of pregnancy (term: 145 days) by dexamethasone injection in 5 animals, while 4 animals were used as control. Elastographic images of the cervix were obtained by two independent operators every 4 hours during 24 hours after injection to monitor the cervical maturation induced by the dexamethasone. Based on the measurements of SWS during vaginal ultrasound examination, the stiffness in the second ring of the cervix was quantified over a circular region of interest of 5 mm diameter. SWS was found to decrease significantly in the first 4–8 hours after dexamethasone compared to controls, which was associated with cervical ripening induced by dexamethasone (from 1.779 m/s ± 0.548 m/s, p < 0.0005, to 1.291 m/s ± 0.516 m/s, p < 0.000). Consequently a drop in the cervical elasticity was quantified too (from 9.5 kPa ± 0.9 kPa, p < 0.0005, to 5.0 kPa ± 0.8 kPa, p < 0.000). Moreover, SWE measurements were highly reproducible between both operators at all times. Cervical ripening induced by dexamethasone was confirmed by the significant increase in maternal plasma Prostaglandin E2 (PGE2), as evidenced by the assay of its metabolite PGEM. Histological analyses and two-photon excitation microscopy, combining both Second Harmonic Generation (SHG) and Two-photon Fluorescence microscopy (2PF) contrasts, were used to investigate, at the microscopic scale, the structure of cervical tissue. Results show that both collagen and 2PF-active fibrillar structures could be closely related to the mechanical properties of cervical tissue that are perceptible in elastography. In conclusion, SWE may be a valuable method to objectively quantify the cervical stiffness and as a complementary diagnostic tool for preterm birth and for labour induction success.     

High-throughput microsatellite isolation through 454 GS-FLX Titanium pyrosequencing of enriched DNA libraries

Microsatellites (or SSRs: simple sequence repeats) are among the most frequently used DNA markers in many areas of research. The use of microsatellite markers is limited by the difficulties involved in their de novo isolation from species for which no genomic resources are available. We describe here a high-throughput method for isolating microsatellite markers based on coupling multiplex microsatellite enrichment and next-generation sequencing on 454 GS-FLX Titanium platforms. The procedure was calibrated on a model species (Apis mellifera) and validated on 13 other species from various taxonomic groups (animals, plants and fungi), including taxa for which severe difficulties were previously encountered using traditional methods. We obtained from 11,497 to 34,483 sequences depending on the species and the number of detected microsatellite loci ranged from 199 to 5791. We thus demonstrated that this procedure can be readily and successfully applied to a large variety of taxonomic groups, at much lower cost than would have been possible with traditional protocols. This method is expected to speed up the acquisition of high-quality genetic markers for nonmodel organisms.     

Towards the establishment of a porcine model to study human amebiasis

Background

Entamoeba histolytica is an important parasite of the human intestine. Its life cycle is monoxenous with two stages: (i) the trophozoite, growing in the intestine and (ii) the cyst corresponding to the dissemination stage. The trophozoite in the intestine can live as a commensal leading to asymptomatic infection or as a tissue invasive form producing mucosal ulcers and liver abscesses. There is no animal model mimicking the whole disease cycle. Most of the biological information on E. histolytica has been obtained from trophozoite adapted to axenic culture. The reproduction of intestinal amebiasis in an animal model is difficult while for liver amebiasis there are well-described rodent models. During this study, we worked on the assessment of pigs as a new potential model to study amebiasis.

Methodology/Principal Findings

We first co-cultured trophozoites of E. histolytica with porcine colonic fragments and observed a disruption of the mucosal architecture. Then, we showed that outbred pigs can be used to reproduce some lesions associated with human amebiasis. A detailed analysis was performed using a washed closed-jejunal loops model. In loops inoculated with virulent amebas a severe acute ulcerative jejunitis was observed with large hemorrhagic lesions 14 days post-inoculation associated with the presence of the trophozoites in the depth of the mucosa in two out four animals. Furthermore, typical large sized hepatic abscesses were observed in the liver of one animal 7 days post-injection in the portal vein and the liver parenchyma.

Conclusions

The pig model could help with simultaneously studying intestinal and extraintestinal lesion development.     

The potential of spatially resolved spectroscopy for monitoring angiogenesis in the chorioallantoic membrane

Over the last decade, the poultry sector has sought to develop ways to monitor chicken embryonic development as to optimize the incubation conditions. One of the parameters of development which may change under different incubation conditions is the angiogenesis in the chorioallantoic membrane (CAM). To be able to quantify these changes in the angiogenesis and detect long-term effects on health, a non-destructive technique is necessary. In this article, the first steps toward such a non-destructive technique are successfully taken. A spatially resolved spectroscopy set-up is built and tested for its potential to measure changes in angiogenesis with incubation time, and differences between a normal and hypercapnic incubation. In this first study, reflectance measurements are performed directly on the CAM as the eggshell considerably complicates the analysis. This issue should be addressed in future research to come to a really non-destructive technique. An experiment was conducted in which one group was incubated under normal conditions, and another under early prenatal hypercapnic conditions (i.e., increased CO(2) concentrations). The angiogenesis in the CAM was measured at embryonic day (ED) 10, 13, and 16. The measurements showed a clear blood spectrum with an increasing amount of blood in time, and significant differences in the reflectance as function of the source-detector distances. However, no significant differences between the hypercapnia and the control group could be detected.     

Kinesin-5/Eg5 is important for transport of CARTS from the trans-Golgi network to the cell surface

Here we report that the kinesin-5 motor Klp61F, which is known for its role in bipolar spindle formation in mitosis, is required for protein transport from the Golgi complex to the cell surface in Drosophila S2 cells. Disrupting the function of its mammalian orthologue, Eg5, in HeLa cells inhibited secretion of a protein called pancreatic adenocarcinoma up-regulated factor (PAUF) but, surprisingly, not the trafficking of vesicular stomatitis virus G protein (VSV-G) to the cell surface. We have previously reported that PAUF is transported from the trans-Golgi network (TGN) to the cell surface in specific carriers called CARTS that exclude VSV-G. Inhibition of Eg5 function did not affect the biogenesis of CARTS; however, their migration was delayed and they accumulated near the Golgi complex. Altogether, our findings reveal a surprising new role of Eg5 in nonmitotic cells in the facilitation of the transport of specific carriers, CARTS, from the TGN to the cell surface.     

Structure of the Tetramerization Domain of Measles Virus Phosphoprotein

The atomic structure of the stable tetramerization domain of the measles virus phosphoprotein shows a tight four-stranded coiled coil. Although at first sight similar to the tetramerization domain of the Sendai virus phosphoprotein, which has a hydrophilic interface, the measles virus domain has kinked helices that have a strongly hydrophobic interface and it lacks the additional N-terminal three helical bundles linking the long helices.     

Gastric intrinsic factor deficiency with combined GIF heterozygous mutations and FUT2 secretor variant

Several genome-wide association studies (GWAS) have identified a strong association between serum vitamin B12 and fucosyltransferase 2 (FUT2), a gene associated with susceptibility to Helicobacter pylori infection. Hazra et al. conducted a meta-analysis of three GWAS and found three additional loci in MUT, CUBN and TCN1. Other GWAS conducted in Italy and China confirmed the association for FUT2 gene. Alpha-2-fucosyltransferase (FUT2) catalyzes fucose addition to form H-type antigens in exocrine secretions. FUT2 non-secretor variant produces no secretion of H-type antigens and is associated with high-plasma vitamin B12 levels. This association was explained by the influence of FUT2 on H. pylori, which is a risk factor of gastritis, a main cause of vitamin B12 impaired absorption. However, we recently showed that H. pylori serology had no influence on FUT2 association with vitamin B12, in a large sample population, suggesting the involvement of an alternative mechanism. GIF is another gene associated with plasma levels of vitamin B12 and gastric intrinsic factor (GIF) is a fucosylated protein needed for B12 absorption. Inherited GIF deficiency produces B12 deficiency unrelated with gastritis. We report 2 families with heterozygous GIF mutation, 290T>C, M97T, with decreased binding affinity of GIF for vitamin B12 and one family with heterozygous GIF mutation 435_437delGAA, K145_N146delinsN and no B12 binding activity of mutated GIF. All cases with vitamin B12 deficit carried the FUT2 rs601338 secretor variant. Ulex europeus binding to GIF was influenced by FUT2 genotypes and GIF concentration was lower, in gastric juice from control subjects with the secretor genotype. GIF290C allele was reported in 5 European cases and no Africans among 1282 ambulatory subjects and was associated with low plasma vitamin B12 and anaemia in the single case bearing the FUT2 secretor variant. We concluded that FUT2 secretor variant worsens B12 status in cases with heterozygous GIF mutations by impairing GIF secretion, independently from H. pylori-related gastritis.