Secondary Metabolites, including a New 5,6-Dihydropyran-2-One, Produced by the Fungus Diplodia corticola. Aphicidal Activity of the Main Metabolite, Sphaeropsidin A.

An undescribed 5,6-dihydropyran-2-one, namely diplopyrone C, was isolated and characterized from the cultures of an isolate of the fungus Diplodia corticola recovered from Quercus suber in Algeria. The structure and relative stereostructure of (5S,6S,7Z,9S,10S)-5-hydroxy-6-(2-(3-methyloxiran-2-yl)vinyl)-5,6-dihydro-2H-pyran-2-one were assigned essentially based on NMR and MS data. Furthermore, ten known compounds were isolated and identified in the same cultures. The most abundant product, the tetracyclic pimarane diterpene sphaeropsidin A, was tested for insecticidal effects against the model sucking aphid, Acyrthosiphon pisum. Results showed a toxic dose-dependent oral activity of sphaeropsidin A, with an LC50 of 9.64 mM.

Targeting the autosomal Ceratitis capitata transformer gene using Cas9 or dCas9 to masculinize XX individuals without inducing mutations.

BACKGROUND: Females of the Mediterranean fruit fly Ceratitis capitata (Medfly) are major agricultural pests, as they lay eggs into the fruit crops of hundreds of plant species. In Medfly, female sex determination is based on the activation of Cctransformer (Cctra). A maternal contribution of Cctra is required to activate Cctra itself in the XX embryos and to start and epigenetically maintain a Cctra positive feedback loop, by female-specific alternative splicing, leading to female development. In XY embryos, the male determining Maleness-on-the-Y gene (MoY) blocks this activation and Cctra produces male-specific transcripts encoding truncated CcTRA isoforms and male differentiation occurs. RESULTS: With the aim of inducing frameshift mutations in the first coding exon to disrupt both female-specific and shorter male-specific CcTRA open reading frames (ORF), we injected Cas9 ribonucleoproteins (Cas9 and single guide RNA, sgRNA) in embryos. As this approach leads to mostly monoallelic mutations, masculinization was expected only in G1 XX individuals carrying biallelic mutations, following crosses of G0 injected individuals. Surprisingly, these injections into XX-only embryos led to G0 adults that included not only XX females but also 50% of reverted fertile XX males. The G0 XX males expressed male-specific Cctra transcripts, suggesting full masculinization. Interestingly, out of six G0 XX males, four displayed the Cctra wild type sequence. This finding suggests that masculinization by Cas9-sgRNA injections was independent from its mutagenic activity. In line with this observation, embryonic targeting of Cctra in XX embryos by a dead Cas9 (enzymatically inactive, dCas9) also favoured a male-specific splicing of Cctra, in both embryos and adults. CONCLUSIONS: Our data suggest that the establishment of Cctra female-specific autoregulation during the early embryogenesis has been repressed in XX embryos by the transient binding of the Cas9-sgRNA on the first exon of the Cctra gene. This hypothesis is supported by the observation that the shift of Cctra splicing from female to male mode is induced also by dCas9. Collectively, the present findings corroborate the idea that a transient embryonic inactivation of Cctra is sufficient for male sex determination.

Simultaneous Silencing of Gut Nucleases and a Vital Target Gene by Adult dsRNA Feeding Enhances RNAi Efficiency and Mortality in Ceratitis capitata.

Ceratitis capitata, known as the Mediterranean fruit fly (Medfly), is a major dipteran pest significantly impacting fruit and vegetable farming. Currently, its control heavily relies mainly on chemical insecticides, which pose health risks and have effects on pollinators. A more sustainable and species-specific alternative strategy may be based on double-stranded RNA (dsRNA) delivery through feeding to disrupt essential functions in pest insects, which is poorly reported in dipteran species. Previous reports in Orthoptera and Coleoptera species suggested that dsRNA degradation by specific nucleases in the intestinal lumen is among the major obstacles to feeding-mediated RNAi in insects. In our study, we experimented with three-day adult feeding using a combination of dsRNA molecules that target the expression of the ATPase vital gene and two intestinal dsRNA nucleases. These dsRNA molecules were recently tested separately in two Tephritidae species, showing limited effectiveness. In contrast, by simultaneously feeding dsRNA against the CcVha68-1, CcdsRNase1, and CcdsRNase2 genes, we observed 79% mortality over seven days, which was associated with a decrease in mRNA levels of the three targeted genes. As expected, we also observed a reduction in dsRNA degradation following RNAi against nucleases. This research illustrates the potential of utilizing molecules as pesticides to achieve mortality rates in Medfly adults by targeting crucial genes and intestinal nucleases. Furthermore, it underscores the importance of exploring RNAi-based approaches for pest management.

Four phases of checks for exclusion of umbilical cord blood donors.

AIM: The aim of this study was to analyse umbilical cord blood (UCB) collection over 1 year between October 2008 and September 2009, seeking ways to improve the number of suitable banked UCB units. Four phases of the process were investigated, from the consent form to the banking procedure, paying attention to the discarded UCB units. MATERIAL AND METHODS: We recruited couples at 35 weeks of gestation and took an accurate history, focusing on genetic, immunological and infectious diseases. We collected UCB from pregnant women who delivered vaginally or by Caesarean section between the 37-41(+6) weeks of gestation. Some units were discarded on the basis of the patients' history, obstetric events or biological criteria. In utero collection was the preferred method of collection. RESULTS: During the study period, between October 2008 and September 2009, there were 1,477 deliveries in our unit. The number of couples interested in UCB donation was 595 (40.2%-595/1,477). We collected 393 UBC units. We excluded 122 patients at the phase of the history taking, counselling and informed consent (first phase check). Of the 393 units collected, 162 (41.3%) were banked whereas 231 (58.7%) were discarded because they did not fulfil biological criteria (third phase check). The volume of UCB units collected after Caesarean section was greater than the volume of units collected after vaginal delivery (95.4 mL versus 85.0 mL, respectively; p <0.01). The UCB units collected after vaginal delivery contained a higher number of total nucleated cells compared to the units collected after Caesarean section (970x10(6) cells versus 874x10(6) cells, respectively; p=0.037). None of the banked UCB units was discarded at the clinical check 6 months after delivery (fourth phase check). CONCLUSIONS: Our study shows that strict observance of each of the checks and the collection strategy is important to guarantee the safety of the UCB units and to maximise the cost-benefit ratio. After the appropriate checks we banked UCB units from only 27.2% (162/595) of the couples who gave consent to the procedure and from only 11% (162/1,477) of all the deliveries in the 12 month study period, as 59.8% of couples were not properly informed about UCB donation.

Prenatal diagnosis of ductus venosus agenesis and its association with cytogenetic/congenital anomalies.

OBJECTIVES: We present an observational study of 12 cases of anomalies of the umbilical and portal vein systems associated with absence of the ductus venosus (DV) diagnosed over the past 5 years. The hemodynamic implications of each pattern of umbilico-portal system anomalies associated with absence of the DV have been investigated, as well as the frequency and types of associated anomalies and their embryological origin. METHODS: In all cases ultrasound, color Doppler, and cytogenetic investigations were performed. RESULTS: Four main patterns of abnormal venous circulation were documented: (1). the umbilical vein (UV) bypasses the liver and drains into the right atrium directly or through a dilated coronary sinus (three cases); (2). the UV bypasses the liver, with an infrahepatic or suprahepatic connection directly to the inferior vena cava (IVC) (two cases); (3). the UV bypasses the liver and drains directly into the iliac or renal veins (four cases); and (4). the UV drains directly into the portal veins (three cases). Among seven cases with other associated anomalies (58%), there were three cases of Turner's and Noonan's syndromes. Two fetuses and two neonates died and there were two terminations of pregnancy (TOP). CONCLUSIONS: In utero diagnosis of ultrasound patterns associated with DV anomalies is feasible. Fetal karyotyping should be considered, serial ultrasound examinations recommended and, in the presence of heart failure, delivery can be anticipated.

22q11 deletions in fetuses with malformations of the outflow tracts or interruption of the aortic arch: impact of additional ultrasound signs.

OBJECTIVES: One hundred and forty-one consecutive cases of malformations of the outflow tracts or interrupted aortic arch (IAA), detected by fetal echocardiography, underwent detailed anatomy scan, karyotyping and fluorescence in situ hybridization analysis (FISH) to detect the prevalence of 22q11 microdeletion and to evaluate neonatal clinical findings and outcome according to the presence of the genetic defect. Then, we sought to investigate whether some prenatal ultrasound findings could help identify fetuses at higher risk of carrying the 22q11 microdeletion. METHODS: Echocardiography and FISH for the DiGeorge critical region (22q11) were performed in all cases. RESULTS: 22q11 microdeletion was detected in 28 of 141 fetuses (19.8%). Intrauterine growth restriction (IUGR) appeared to be associated with the worst prognosis, being present in 2/2 intrauterine fetal deaths and 5/6 post-natal deaths. IUGR, additional aortic arch anomalies and thymic hypo/aplasia were significantly more frequent in fetuses with 22q11 microdeletion (p=0.011, 0.011 and <0.0001, respectively). Prenatal ultrasound thymus examination, performed on the last 84 fetuses, showed 75% sensitivity and 94% specificity. The combination of 2 predictors, namely, thymus defects and IUGR associated with additional aortic arch anomalies reached more than 90% sensitivity and 100% specificity. CONCLUSIONS: Our study demonstrates that 22q11 microdeletion occurs in 20% of malformations of the outflow tracts and IAA type B, as detected in utero, and that this association is significantly predicted by the presence of associated ultrasound findings: thymic hypo/aplasia, IUGR and additional aortic arch anomalies. The feasibility of a correct prenatal diagnosis should enable clinicians to provide the couple with further informative counselling and to plan adequate post-natal medical interventions.