RUS6, a DUF647-containing protein, is essential for early embryonic development in Arabidopsis thaliana

Background The Arabidopsis RUS (ROOT UV-B SENSITIVE) gene family contains six members, each of which encodes a protein containing a DUF647 (domain of unknown function 647) that is commonly found in eukaryotes. Previous studies have demonstrated that RUS1 and RUS2 play critical roles in early seedling development. All six RUS genes are expressed throughout the plant, but little is known about the functional roles of RUS3, RUS4, RUS5 and RUS6. Results We used a reverse-genetic approach to identify knockout mutants for RUS3, RUS4, RUS5 and RUS6. Each mutant was confirmed by direct DNA sequencing and genetic segregation analysis. No visible phenotypic differences were observed in rus3, rus4, or rus5 knockout mutants under standard growth conditions, but rus6 knockout mutants displayed a strong embryo-lethal phenotype. Two independent knockout lines for RUS6 were characterized. The rus6 mutations could only be maintained through a heterozygote, because rus6 homozygous mutants did not survive. Closer examinations of homozygous rus6 embryos from rus6/ + parent plants revealed that RUS6 is required for early embryo development. Loss of RUS6 resulted in embryo lethality, specifically at the mid-globular stage. The embryo-lethality phenotype was complemented by a RUS6::RUS6-GFP transgene, and GFP signal was detected throughout the embryo. Histological analyses with the β-glucuronidase reporter gene driven by the RUS6 promoter showed tissue- and development-specific expression of RUS6, which was highest in floral tissues. Conclusion Our data revealed that RUS6 is essential for early embryo development in Arabidopsis, and that the RUS gene family functions in multiple stages of plant development.

Embryo Lethality Assay for Evaluating Virulence of Isolates from Bacterial Chondronecrosis with Osteomyelitis in Broilers

We used an embryo lethality assay (ELA) to assess virulence for different isolates from cases of bacterial chondronecrosis with osteomyelitis (BCO) in broilers. ELA has been used to measure virulence and lethal dosage of Enterococcus faecalis and Enterococcus cecorum. We hypothesized that ELA could substitute for more laborious and costly assessments of BCO isolate pathogenicity using live birds. We evaluated two different levels of bacteria injected into eggs from layer and commercial broiler embryos. Significant findings include a) Escherichia coli from neighboring farms operated by the same integrator had very different embryo lethality, b) isolate Staphylococcus agnetis 908 had low virulence in ELA, even though this isolate can induce more than 50% BCO lameness, c) Enterococcus cecorum 1415 also had low pathogenicity; even though it was recovered from severe bilateral tibial dyschondroplasia, d) human and chicken isolates of S. aureus had significant pathogenicity, e) virulence for some isolates was highly variable possibly corresponding with quality of the embryos/fertile eggs used, and f) ELA pathogenicity was much lower for our BCO isolates than previous reports which may reflect maternal environment. Overall, ELA virulence and BCO virulence are not always concordant indicating that that ELA may not be an effective measure for assessing virulence with respect to BCO.ImportanceLameness is among the most significant animal welfare issues in the poultry industry. Bacterial infections are a major cause of lameness and different bacterial species have been obtained from lame broilers. Reliable lab-based assays are required to assess relative virulence of bacteria obtained from lame broilers. Embryo Lethality Assays have been used to compare virulence. Our results suggest that this assay may not be an effective measure of virulence related to lameness.

RUS6, a DUF647-Containing Protein, is Essential for Early Embryonic Development in Arabidopsis Thaliana

BackgroundThe Arabidopsis RUS (ROOT UV-B SENSITIVE) gene family contains six members, each of which encodes a protein containing a DUF647 (domain of unknown function 647) that is commonly found in eukaryotes. Previous studies have demonstrated that RUS1 and RUS2 play critical roles in early seedling development. All six RUS genes are expressed throughout the plant, but little is known about the functional roles of RUS3, RUS4, RUS5 and RUS6. ResultsWe used a reverse-genetic approach to identify knockout mutants for RUS3, RUS4, RUS5 and RUS6. Each mutant was confirmed by direct DNA sequencing and genetic segregation analysis. No visible phenotypic differences were observed in rus3, rus4, or rus5 knockout mutants under standard growth conditions, but rus6 knockout mutants displayed a strong embryo-lethal phenotype. Two independent knockout lines for RUS6 were characterized. The rus6 mutations could only be maintained through a heterozygote, because rus6 homozygous mutants did not survive. Closer examinations of homozygous rus6 embryos from rus6/+ parent plants revealed that RUS6 is required for early embryo development. Loss of RUS6 resulted in embryo lethality, specifically at the mid-globular stage. The embryo-lethality phenotype was complemented by a RUS6::RUS6-GFP transgene, and GFP signal was detected throughout the embryo. Histological analyses with the β-glucuronidase reporter gene driven by the RUS6 promoter showed tissue- and development-specific expression of RUS6, which was highest in floral tissues. ConclusionOur data revealed that RUS6 is essential for early embryo development in Arabidopsis, and that the RUS gene family functions in multiple stages of plant development.

Virulence and antibiotic resistance profile of avian Escherichia coli strains isolated from colibacillosis lesions in central of Algeria

Background and Aim: Avian pathogenic Escherichia coli cause extensive mortality in poultry flocks, leading to extensive economic losses. To date, in Algeria, little information has been available on virulence potential and antibiotics resistance of avian E. coli isolates. Therefore, the aim of this study was the characterization of virulence genes and antibiotic resistance profile of Algerian E. coli strains isolated from diseased broilers. Materials and Methods: In this study, 43 avian E. coli strains isolated from chicken colibacillosis lesions at different years were analyzed to determine their contents in 10 virulence factors by polymerase chain reaction, antimicrobial susceptibility to 22 antibiotics belonging to six different chemical classes and genomic diversity by pulsed-field gel electrophoresis (PFGE). Results: Mainly E. coli isolates (58.1%) carried two at six virulence genes and the most frequent virulence gene association detected were ompT (protectin), hlyF (hemolysin) with 55.8% (p<0.001), and iroN, sitA (iron acquisition/uptake systems), and iss (protectin) with 41.8% (p<0.001). Some strains were diagnosed as virulent according to their virulence gene profile. Indeed, 23.25% of the isolates harbored iroN, ompT, hlyF, iss, and sitA combination, 14% ompT, hlyF, and frzorf4 (sugar metabolism), and 11,6% iroN, hlyF, ompT, iss, iutA (iron acquisition/uptake systems), and frzorf4. The chicken embryo lethality assay performed on five isolates confirmed the potential virulence of these strains. All isolates submitted to PFGE analysis yielded different genetic profiles, which revealed their diversity. Overall, 97.2% of the isolates were resistant to at least one antibiotic and 53.5% demonstrated multi-antimicrobial resistance to three different antimicrobial classes. The highest resistance levels were against nalidixic acid (83.4%), amoxicillin and ampicillin (83.3%), ticarcillin (80.5%), pipemidic acid (75%), and triméthoprim-sulfamethoxazole (66.6%). For beta-lactam class, the main phenotype observed belonged to broad-spectrum beta-lactamases. However, extended-spectrum beta-lactamase associated with three at six virulence factors was also detected in 13 isolates. Two of them were attested virulent as demonstrated in the embryo lethality test which constitutes a real public threat. Conclusion: It would be imperative in avian production to discourage misuse while maintaining constant vigilance guidelines and regulations, to limit and rationalize antimicrobial use.

Presence of Virulence Genes in Enterococcus Species Isolated from Meat Turkeys in Germany Does Not Correlate with Chicken Embryo Lethality

Virulence-associated traits have frequently been studied in enterococci and are considered to contribute towards the pathogenicity of infections. In the present study, Enterococcus isolates were collected during diagnostic investigations from meat turkeys in Germany. Twenty-eight isolates of three different Enterococcus species were analyzed for five selected putative virulence traits to understand their potential role in the pathogenicity using the chicken embryo lethality assay. Ten E. faecalis, ten E. faecium, and eight E. gallinarum isolates were examined for the presence of common virulence genes and their phenotypic expression, namely, the cytolysin operon, five individual cyl genes (cylLL, cylLS, cylM, cylB, and cylA), gelatinase (gelE), hyaluronidase (hylEfm), aggregation substance (asa1), and enterococcal surface protein (esp). The Enterococcus isolates showed significant species-dependent differences in the presence of genotypic traits (p<0.001 by Fisher’s exact test; Cramer’s V = 0.68). At least one gene and up to three virulence traits were found in E. faecalis, while six E. faecium isolates and one E. gallinarum isolate did not display any virulence-associated pheno- or genotype. More than half of the Enterococcus isolates (n = 15) harbored the gelE gene, but only E. faecalis (n = 10) expressed the gelatinase activity in vitro. The hylEfm gene was found in five E. gallinarum isolates only, while seven isolates showed the hyaluronidase activity in the phenotypic assay. In Cramer’s V statistic, a moderate association was indicated for species (V ≤ 0.35) or genotype (V < 0.43) and the results from the embryo lethality assay, but the differences were not significant. All E. gallinarum isolates were less virulent with mortality rates ranging between 0 and 30%. Two E. faecalis isolates were highly virulent, harboring the whole cyl-operon as well as gelE and asa1 genes. Likewise, one E. faecium isolate caused high embryo mortality but did not harbor any of the investigated virulence genes. For the first time, Enterococcus isolates of three different species collected from diseased turkeys were investigated for their virulence properties in comparison. The results differed markedly between the Enterococcus species, with E. faecalis harboring the majority of investigated genes and virulence traits. However, the genotype did not entirely correlate with the phenotype or the isolates’ virulence potential and pathogenicity for chicken embryos.

Cryptolepine, the Main Alkaloid of the Antimalarial Cryptolepis sanguinolenta (Lindl.) Schlechter, Induces Malformations in Zebrafish Embryos

Background. Previous studies on cryptolepine, the antimalarial and cytotoxic alkaloid of Cryptolepis sanguinolenta, showed that it preferentially accumulates in rapidly proliferating cells and melanin-containing tissues. Subsequently, we demonstrated that cryptolepine was toxic to murine embryos in vivo but no signs of teratogenicity. in vivo developmental studies can be confounded by maternal effects. Here, we hypothesized that cryptolepine-induced embryo toxicity occurs at least partly through direct inhibition of embryogenesis rather than indirectly through the induction of maternal toxicity. Aim. To determine the effects of cryptolepine on developing zebrafish embryos ex vivo. Methods. Healthy synchronized zebrafish eggs were treated with cryptolepine (10−1 − 5 × 102 μM), benzyl penicillin (6 − 6 × 102 μM), or mercury chloride (3.7 × 10−1 − 3.7 × 101 nM) from 6 to 72 hours postfertilization. Developing embryos were assessed at 24, 48, 72, and 96 hours under microscope for lethality, hatching rate, and malformation. Results. LC50 for cryptolepine in the study was found to be 260 ± 0.174 μM. Cryptolepine induced dose- and time-dependent mortality from the 24 to 96 hours postfertilization. Lower cryptolepine concentration (<100 μM) caused mortality, approximately 15–18%, only after the 48 hours postfertilization. The most sensitive period of embryo lethality corresponded well with the pharyngula (24 to 48 hours) and hatching (48 to 72 hours) stages of embryonic development. Cryptolepine (10−1 − 5 × 102 μM) dose dependently inhibited the hatching rate. At doses above 500 μM, hatching was completely inhibited. Mercury chloride (3.7 × 10−1 − 3.7 × 101 nM), used as positive control, induced a consistent pattern of embryo lethality at all stages of development, whereas benzyl penicillin (6 − 6 × 102 μM), used as negative control, did not induce any significant embryo lethality. Morphological examination of (postfertilization day 5) of eleutheroembryos treated during embryonic development with cryptolepine showed decreased body length (growth inhibition), decreased eye diameter and bulginess, enlarged pericardia, and enlarged yolk sac and muscle malformations. Conclusion. Cryptolepine induces malformations, growth retardation, and mortalities in rapidly dividing zebrafish embryos ex vivo.