Generation of Mycoplasma hominis gene-targeted mutants by targeting-induced local lesions in genomes (TILLING).

BACKGROUND: Mycoplasma hominis is a human urogenital pathogen involved in gynaecological, neonatal and extra-genital infections. However, no versatile genetic tools are currently available to study the pathogenicity of this bacterium. Targeting-Induced Local Lesions IN Genomes (TILLING) is a reverse-genetic method that combines point mutations induced by chemical mutagenesis with a DNA screening technique. We used ethyl methanesulfonate (EMS) that introduces C-G to T-A transition mutations to generate a library of M. hominis mutants. As a proof of concept, mutagenized organisms were screened for mutations in two target genes previously associated with the mycoplasma pathogenicity, the vaa gene encoding an adhesin lipoprotein and the oppA gene encoding the main ectoATPase of the bacterium. The resulting mutants were evaluated using functional assays, an adhesion to HeLa cell assay for vaa-mutants and an ATPase activity test for oppA-mutants. RESULTS: A 1200-clone library was generated by exposing M. hominis PG21 to 9 mg/mL EMS for 3 h. To identify mutants of interest, targeted gene fragments were amplified, heat-denatured, slowly reannealed and digested with the mismatch-specific endonuclease ENDO1. If multiple alleles were present in the PCR amplicons, these alleles formed heteroduplexes during reannealing that were specifically cleaved by ENDO1 at mismatching positions. A total of four vaa-mutants and two oppA-mutants harbouring missense mutations were obtained and fully sequenced. Zero to eight additional mutations were identified in the genomes of each mutant. The vaa-mutants were tested for adhesion to immobilized HeLa cells but their adhesion was not significantly different from the adhesion of M. hominis PG21. One of the two oppA-mutants that were tested for ATPase activity presented a higher affinity for its ATP substrate than the parental strain. CONCLUSION: For the first time, we demonstrated that M. hominis gene-targeted mutants could be successfully obtained using this TILLING strategy. In the absence of robust genetic tools for studying M. hominis, the TILLING strategy that can target any gene of the genome could help to elucidate gene functions and to better understand the pathogenesis of this human pathogenic species.

Disruption of an ant-plant mutualism shapes interactions between lions and their primary prey.

Mutualisms often define ecosystems, but they are susceptible to human activities. Combining experiments, animal tracking, and mortality investigations, we show that the invasive big-headed ant (Pheidole megacephala) makes lions (Panthera leo) less effective at killing their primary prey, plains zebra (Equus quagga). Big-headed ants disrupted the mutualism between native ants (Crematogaster spp.) and the dominant whistling-thorn tree (Vachellia drepanolobium), rendering trees vulnerable to elephant (Loxodonta africana) browsing and resulting in landscapes with higher visibility. Although zebra kills were significantly less likely to occur in higher-visibility, invaded areas, lion numbers did not decline since the onset of the invasion, likely because of prey-switching to African buffalo (Syncerus caffer). We show that by controlling biophysical structure across landscapes, a tiny invader reconfigured predator-prey dynamics among iconic species.

Dexmedetomidine sedation vs. inhaled general anesthesia for pediatric MRI: A retrospective cohort study: Dexmedetomidine sedation vs. inhaled general anesthesia for MRI.

OBJECTIVES: The objective of this study was to evaluate the feasibility and the efficacy of a dexmedetomidine-based protocol followed by anesthesiologists unaccustomed to using dexmedetomidine during pediatric magnetic resonance imaging (MRI) examinations compared to conventional halogenated general anesthesia. METHODS: This was a single-center retrospective cohort study including patients younger than 18 years who underwent sedation for MRI between August 1, 2018 and March 31, 2019. Patients who received dexmedetomidine were included in the DEX group and patients who had general anesthesia formed the GA group. Patients were matched with a ratio of 2 GA:1 DEX, based on age and type of MRI examination. RESULTS: Overall, 78 patients were included (DEX=26; GA=52). Dexmedetomidine was significantly associated with a decrease in invasive ventilation (p<0.001) with no impact on image quality. The sedation failure rate was 42% with dexmedetomidine vs. 0% with general anesthesia (p<0.001). All cases of failure followed the intranasal administration of dexmedetomidine. CONCLUSION: Dexmedetomidine seems to be a suitable sedation option for pediatric MRI. It provides an alternative to halogenated general anesthesia with the aim of limiting exposure to conventional anesthetic agents and invasive ventilation.

Demographic consequences of mutualism disruption: Browsing and big-headed ant invasion drive acacia population declines.

Across the globe, biological invasions have disrupted mutualisms, producing reverberating consequences for ecosystems. Although invasive species frequently trigger mutualism disruptions, few studies have quantified the demographic mechanisms by which mutualism breakdown may generate population effects. In a Kenyan savanna, the invasive big-headed ant (Pheidole megacephala) has disrupted a foundational mutualism between the monodominant whistling-thorn tree (Acacia drepanolobium) and native ants (Crematogaster spp.) that deter browsing by large mammalian herbivores. We conducted experiments to quantify the demographic consequences of this mutualism disruption in the presence and absence of large mammalian herbivores. Invasion by P. megacephala exacerbated population declines of A. drepanolobium, primarily through decreased survival and reproduction of adult trees. However, these fitness reductions were small compared to those resulting from the presence of large mammalian herbivores, which negatively impacted growth and survival. Contrary to expectation, the expulsion of metabolically costly Crematogaster mutualists by P. megacephala did not result in higher population growth rates for trees protected from large mammalian herbivores. Our results suggest that invasive P. megacephala may impose a direct metabolic cost to trees exceeding that of native mutualists while providing no protection from browsing by large mammalian herbivores. Across landscapes, we expect that invasion by P. megacephala will reduce A. drepanolobium populations, but that the magnitude and demographic pathways of this effect will hinge on the presence and abundance of browsers.

Fetal tumors of the choroid plexus: is differential diagnosis between papilloma and carcinoma possible?

Fetal choroid plexus tumors are uncommon. The prognosis is widely variable and depends on the histological findings: papilloma or carcinoma. We report a case of prenatal diagnosis of choroid plexus mass detected by ultrasound at 33 weeks of gestation. Prenatal (T1, T2, T2* and diffusion weighted sequences) magnetic resonance imaging (MRI) was used to rule out a hematoma. Follow-up examination by ultrasound and MRI revealed a significant increase in the volume of the mass, suggesting a diagnosis of malignant tumor. A healthy neonate was delivered by Cesarean section at 38 weeks of gestation. Full surgical excision of the tumor was performed at 20 days after delivery and histological analysis revealed a papilloma.

Genetics of lifespan in C. elegans: molecular diversity, physiological complexity, mechanistic simplicity.

The nematode Caenorhabditis elegans is used as a model system for the study of aging. Several mutant strains that have an increased lifespan have been isolated and characterized genetically and molecularly. Molecular analysis reveals that diverse types of gene products can affect worm lifespan, including proteins active in signal transduction, transcription and silencing factors, mitochondrial enzymes, and at least one protein that affects telomere length. Genetic analysis, however, suggests that these activities all converge on a few key mechanisms that impinge on lifespan, namely the production, repair and prevention of molecular damage.

Basolateral localization of the Caenorhabditis elegans epidermal growth factor receptor in epithelial cells by the PDZ protein LIN-10.

In Caenorhabditis elegans, the EGF receptor (encoded by let-23) is localized to the basolateral membrane domain of the epithelial vulval precursor cells, where it acts through a conserved Ras/MAP kinase signaling pathway to induce vulval differentiation. lin-10 acts in LET-23 receptor tyrosine kinase basolateral localization, because lin-10 mutations result in mislocalization of LET-23 to the apical membrane domain and cause a signaling defective (vulvaless) phenotype. We demonstrate that the previous molecular identification of lin-10 was incorrect, and we identify a new gene corresponding to the lin-10 genetic locus. lin-10 encodes a protein with regions of similarity to mammalian X11/mint proteins, containing a phosphotyrosine-binding and two PDZ domains. A nonsense lin-10 allele that truncates both PDZ domains only partially reduces lin-10 gene activity, suggesting that these protein interaction domains are not essential for LIN-10 function in vulval induction. Immunocytochemical experiments show that LIN-10 is expressed in vulval epithelial cells and in neurons. LIN-10 is present at low levels in the cytoplasm and at the plasma membrane and at high levels at or near the Golgi. LIN-10 may function in secretion of LET-23 to the basolateral membrane domain, or it may be involved in tethering LET-23 at the basolateral plasma membrane once it is secreted.

Why only time will tell.

The nematode Caenorhabditis elegans has become a model system for the study of the genetic basis of aging. In particular, many mutations that extend life span have been identified in this organism. When loss-of-function mutations in a gene lead to life span extension, it is a necessary conclusion that the gene normally limits life span in the wild type. The effect of a given mutation depends on a number of environmental and genetic conditions. For example, the combination of two mutations can result in additive, synergistic, subtractive, or epistatic effects on life span. Valuable insight into the processes that determine life span can be obtained from such genetic analyses, especially when interpreted with caution, and when molecular information about the interacting genes is available. Thus, genetic and molecular analyses have implicated several genes classes (daf, clk and eat) in life span determination and have indicated that aging is affected by alteration of several biological processes, namely dormancy, physiological rates, food intake, and reproduction.

[Arterial hypertension in pregnancy. Role of maternal and fetal blood velocimetry]. / Hypertension artérielle gravidique. Place de la vélocimétrie sanguine maternelle et foetale.

Gravidic hypertension remains one of the most frequent causes of perinatal mortality and morbidity. There are two aims when investigating this disease: evaluate the gravity, try to find early signs of a risk of pre-eclampsia. This last point has become particularly important over the past few years because of the development of preventive treatment (platelet anti-agregates). Mother and fetal blood velocimetry play a more and more important role. Three measurement sites appear to have different and complementary importance: for long-term prediction, measuring the velocimetric index of the uterin artery seems the most interesting because it schematically explores the type of placentations; for mid-term, measuring the ombilical artery evaluates placenta resistance, an essential factor in chronic fetal suffering; at short-term, measuring the fetal cerebral and carotid vessels explores the hemodynamic reactions of fetal adaptation to fetal suffering.

clk-1, mitochondria, and physiological rates.

Mutations in the C. elegans maternal-effect gene clk-1 are highly pleiotropic, affecting the duration of diverse developmental and behavioral processes. They result in an average slowing of embryonic and post-embryonic development, adult rhythmic behaviors, reproduction, and aging.(1) CLK-1 is a highly conserved mitochondrial protein,(2,3) but even severe clk-1 mutations affect mitochondrial respiration only slightly.(3) Here, we review the evidence supporting the regulatory role of clk-1 in physiological timing. We also discuss possible models for the action of CLK-1, in particular, one proposing that CLK-1 is involved in the coordination of mitochondrial and nuclear function. BioEssays 22:48-56, 2000.

Treatment of rat proximal and distal colonic cells with sodium orthovanadate enhances their adhesion and survival in primary culture.

We have studied the effect of sodium orthovanadate, an inhibitor of protein tyrosine phosphatases, on primary cultures of colonocytes and stromal cells. Everted proximal and distal colonic tissue of adult rats were disintegrated by a collagenase/dispase solution for 60 min at 37 degrees C to prepare viable gland fragments and isolated cells. Cell preparations were inoculated onto plastic substratum or cytodex-3 microcarriers in a defined maintenance medium or in 1% fetal calf serum media. Incorporation of sodium orthovanadate (> or = 50 microM) in these media constantly enhanced the survival (cell enumeration and trypan blue exclusion P < 0.05) and the adhesion (up to four-fold by crystal violet staining, P < 0.01) of colonocytes (characterized by cytokeratin-18, transforming growth factor-alpha or alkaline phosphatase expression) and stromal cells. Removal of sodium orthovanadate from culture media restored cellular death processes. Incorporation of 10 mM n-butyric acid did not promote cell adhesion and survival except for distal cells exposed to 2 mM sodium orthovanadate. Besides studies in the regulation of anoikis in primary culture, the model will help to assay the influences of dietary and growth factors on the biology of non-cancerous colonic cells.

In utero fetal cerebral intraparenchymal hemorrhage associated with an abnormal cerebral Doppler.

Cerebral hemorrhages are the most serious complication in preterm and hypotrophic neonates. The mechanisms underlying the formation of these hemorrhages are unclear. In utero Doppler cerebral velocimetry is not yet used as a parameter to predict this complication. Our case allowed us to analyze the evolution of cerebral Doppler abnormalities with the appearance of an intracerebral hemorrhage. The modifications in the Doppler cerebral velocimetry could not be predicted. Therefore it is necessary to determine other parameters which show the evolution of the intracerebral vasomotricity.

Biological properties of ulvan, a new source of green seaweed sulfated polysaccharides, on cultured normal and cancerous colonic epithelial cells.

Ulvans (from Ulva lactuca) constitute a dietary fiber structurally similar to the mammalian glycosaminoglycans but with unexplored biological or cytotoxic activities. From native low-viscosity preparations containing 33.5 molar % and 18.4 molar % of sulfate residues and uronic acid residues, respectively, we derived desulfated, reduced and desulfated-reduced polysaccharides with respectively 5.2, 2.9, and 4.5-4.9 molar % of sulfate residues and uronic acid residues. The effects of these preparations were examined on the adhesion, proliferation and differentiation of normal or tumoral colonic epithelial cells cultured in conventional (0.3-0.8 x 10(6) cells/ml) or rotating bioreactor (3-8 x 10(6) cells/ml) culture conditions. In conventional culture conditions, ulvan modified the adhesion phase and the proliferation of normal colonic cells and undifferentiated HT-29 cells according to their molecular weights and to the relative molar proportion of sulfate residues. From the native polysaccharides, we have screened sulfated ulvans (MW < 5,000) which inhibited the Caco-2 cell proliferation/differentiation program by inducing a low cell reactivity to Ulex europeaus-1 lectins in defined (p < 0.001) or serum-supplemented media (p < 0.01) but were inactive on normal colonocytes. In conclusion, this dietary fiber could be a source of oligosaccharides with a bioactivity, a cytotoxicity or a cytostaticity targeted to normal or cancerous epithelial cells.

Relationship between nuchal translucency at 11-14 weeks and nuchal fold at 20-24 weeks of gestation.

OBJECTIVES: To examine the relationship between the measurement of nuchal translucency in the first trimester and nuchal fold in the second trimester in normal pregnancy. METHODS: This was a prospective study of 592 singleton pregnancies. Fetal nuchal translucency was measured at 11-14 weeks of gestation and nuchal fold at 20-24 weeks of gestation. Linear regression models were used to assess the relationship between nuchal translucency and nuchal fold after adjustment for gestational age. RESULTS: There was no significant association between nuchal translucency and nuchal fold thickness. CONCLUSION: It is possible that measurement of nuchal translucency and nuchal fold may provide an independent contribution in screening for trisomy 21.

The C. elegans maternal-effect gene clk-2 is essential for embryonic development, encodes a protein homologous to yeast Tel2p and affects telomere length.

The Caenorhabditis elegans maternal-effect clk genes are involved in the temporal control of development and behavior. We report the genetic and molecular characterization of clk-2. A temperature-sensitive mutation in the gene clk-2 affects embryonic and post-embryonic development, reproduction, and rhythmic behaviors. Yet, virtually all phenotypes are fully maternally rescued. Embryonic development strictly requires the activity of maternal clk-2 during a narrow time window between oocyte maturation and the two- to four-cell embryonic stage. Positional cloning of clk-2 reveals that it encodes a protein homologous to S. cerevisiae Tel2p. In yeast, the gene TEL2 regulates telomere length and participates in gene silencing at subtelomeric regions. In C. elegans, clk-2 mutants have elongated telomeres, and clk-2 overexpression can lead to telomere shortening. Tel2p has been reported to bind to telomeric DNA repeats in vitro. However, we find that a functional CLK-2::GFP fusion protein is cytoplasmic in worms. We discuss how the phenotype of clk-2 mutants could be the result of altered patterns of gene expression.

Cellular and axonal migrations are misguided along both body axes in the maternal-effect mau-2 mutants of Caenorhabditis elegans.

We have characterized the mau-2 mutants of Caenorhabditis elegans and found that migrating cells and axons are mispositioned along both the antero-posterior and dorsoventral body axes. This is in contrast to previously characterized guidance mutations in Caenorhabditis and in Drosophila, which have been found to be axis-specific. Two observations suggest that mau-2 acts very early during development: most behavioral phenotypes of mau-2 can be rescued by a maternal effect, and variations in expressivity involve an entire body side at a time. The possibility that mau-2 is involved in the spatial organization of guidance cues encoded by other genes is discussed.

Relationship between umbilical and fetal cerebral blood flow velocity waveforms and umbilical venous blood gases.

Nineteen women (mean +/- SD: 28 +/- 5 years) with small fetuses relative to gestational age (mean +/- SD: 32 +/- 2.2 weeks of gestation) were referred to our unit for cordocentesis for the determination of fetal karyotyping. Four of these 19 patients had small-for-gestational-age ultrasound-derived measurements under the third centile for gestational age. Fetal blood gases were measured in the umbilical vein. Umbilical artery and fetal internal carotid artery Doppler examinations were performed 15 min before cordocentesis. The results showed a progressive and proportional increase in umbilical artery resistance index with hypoxia (p < 0.001), hypercapnia (p < 0.025) and acidosis (p < 0.005). Moreover, the results showed a progressive and proportional decrease in fetal internal carotid artery resistance index with hypoxia (p < 0.01), hypercapnia (p < 0.01) and acidosis (p < 0.025). Fetal blood gases are therefore related to umbilical circulatory function and cerebral vasodilatation, suggesting fetal vascular redistribution.