First steps towards Echinometra lucunter embryo cryopreservation

We have studied the sensitivity to cryoprotecting agents of different embryos of the local sea urchin, Echinometra lucunter which is the species used for embryo-larval bioassays in Brazil. We have located significant differences between both species sensitivity to cryoprotecting agents; while for P. lividus propylene glycol was the less toxic compound for most development stages, whereas for E. lucunter is was the most toxic and the least toxic was Dimethyl sulfoxide. There is a significant difference between development stages as well; in the case of P. lividus, the blastula embryo was the most resistant to the cryoprotecting agents, meanwhile for E. lucunter, it was the fertilized oocyte. This is a very promising result, a really early embryo that is not extremely sensitive to Me₂SO. Our next aim is to develop a cryopreservation protocol for E. lucunter early embryos and test them in an embryo-larval bioassay.     

DNA modification and functional delivery into human cells using Escherichia coli DH10B

The availability of almost the complete human genome as cloned BAC libraries represents a valuable resource for functional genomic analysis, which, however, has been somewhat limited by the ability to modify and transfer this DNA into mammalian cells intact. Here we report a novel comprehensive Escherichia coli-based vector system for the modification, propagation and delivery of large human genomic BAC clones into mammalian cells. The GET recombination inducible homologous recombination system was used in the BAC host strain E.coli DH10B to precisely insert an EGFPneo cassette into the vector portion of a ~200 kb human BAC clone, providing a relatively simple method to directly convert available BAC clones into suitable vectors for mammalian cells. GET recombination was also used for the targeted deletion of the asd gene from the E.coli chromosome, resulting in defective cell wall synthesis and diaminopimelic acid auxotrophy. Transfer of the Yersinia pseudotuberculosis invasin gene into E.coli DH10B asd^– rendered it competent to invade HeLa cells and deliver DNA, as judged by transient expression of green fluorescent protein and stable neomycin-resistant colonies. The efficiency of DNA transfer and survival of HeLa cells has been optimized for incubation time and multiplicity of infection of invasive E.coli with HeLa cells. This combination of E.coli-based homologous recombination and invasion technologies using BAC host strain E.coli DH10B will greatly improve the utility of the available BAC libraries from the human and other genomes for gene expression and functional genomic studies.     

Profound Activity of the Anti-cancer Drug Bortezomib against Echinococcus multilocularis Metacestodes Identifies the Proteasome as a Novel Drug Target for Cestodes

A library of 426 FDA-approved drugs was screened for in vitro activity against E. multilocularis metacestodes employing the phosphoglucose isomerase (PGI) assay. Initial screening at 20 µM revealed that 7 drugs induced considerable metacestode damage, and further dose-response studies revealed that bortezomib (BTZ), a proteasome inhibitor developed for the chemotherapy of myeloma, displayed high anti-metacestodal activity with an EC₅₀ of 0.6 µM. BTZ treatment of E. multilocularis metacestodes led to an accumulation of ubiquinated proteins and unequivocally parasite death. In-gel zymography assays using E. multilocularis extracts demonstrated BTZ-mediated inhibition of protease activity in a band of approximately 23 kDa, the same size at which the proteasome subunit beta 5 of E. multilocularis could be detected by Western blot. Balb/c mice experimentally infected with E. multilocularis metacestodes were used to assess BTZ treatment, starting at 6 weeks post-infection by intraperitoneal injection of BTZ. This treatment led to reduced parasite weight, but to a degree that was not statistically significant, and it induced adverse effects such as diarrhea and neurological symptoms. In conclusion, the proteasome was identified as a drug target in E. multilocularis metacestodes that can be efficiently inhibited by BTZ in vitro. However, translation of these findings into in vivo efficacy requires further adjustments of treatment regimens using BTZ, or possibly other proteasome inhibitors.     

Genetic studies to re-affiliate Edwardsiella tarda fish isolates to Edwardsiella piscicida and Edwardsiella anguillarum species

Until 2012, the genus Edwardsiella was composed by three species Edwardsiella tarda, Edwardsiella hoshinae and Edwardsiella ictaluri. In 2013, Edwardsiella piscicida, compiling fish pathogenic strains previously identified as E. tarda was described, and more recently a new species isolated from diseased eel was reported, namely Edwardsiella anguillarum.The incorporation of these species into the genus makes necessary a revision of the taxonomic position of the isolates previously identified as E. tarda. Using AFLP technique, MLSA studies and in silico DNA–DNA hybridization, 46 of 49 E. tarda isolates were re-assigned as E. piscicida and 2 as E. anguillarum, whereas it was confirmed previous classification of the Edwardsiella types and reference strains used. The study of the taxonomic resolution of the genes 16S rRNA, adk, atpD, dnaJ, glnA, hsp60, tuf as well as the possible combinations among housekeeping genes, showed that the gene dnaJ was the more resolutive. In conclusion, the use of molecular techniques is necessary to accurately identify Edwardsiella isolates, especially when differentiating new species from E. tarda.     

Ensifer adhaerens Predatory Activity Against Other Bacteria in Soil, as Monitored by Indirect Phage Analysis

An indirect phage analysis procedure was used to detect and follow the activity of the bacterial predator Ensifer adhaerens in situ in natural soil. The soil was percolated with an aqueous suspension of washed bacterial host cells so that the E. adhaerens cells naturally present in the soil would multiply in response to the host cells. The natural phage development which ensued against these multiplying E. adhaerens cells in the soil was then monitored by noting plaques which developed when the percolation fluid was plated with laboratory strains of E. adhaerens on laboratory media. The activities of the other members of the predation system that includes E. adhaerens (Streptomyces sp. strain 34 and a myxobacter) could not be monitored directly by phage analysis because phage were not found for them. Indirect monitoring was possible, however, because they were susceptible to attack by E. adhaerens. In general, the results were in agreement with previous observations by other methods of the predation sequence. E. adhaerens attacked Micrococcus luteus, Streptomyces sp. strain 34, and the myxobacter but did not attack several other possible species of hosts. It also did not respond to percolation of the soil with various nutrient solutions. E. adhaerens phage activity was not present in half of the soils percolated with M. luteus cells. This seemed to reflect too great a phage-host specificity for the technique as regards these soils, because E. adhaerens-like bacteria other than the strains used for plaquing were present in at least some of these soils. Although E. adhaerens did not attack Escherichia coli or Pseudomonas aeruginosa in soil, there was an overproduction of E. adhaerens phage if these bacteria were percolated simultaneously with M. luteus cells. The possibility is discussed that this represents an activation by M. luteus (or by a heat-extractable factor from it) of other bacterial predators that attack E. coli or P. aeruginosa and that these predators subsequently are themselves attacked by E. adhaerens.     

Adherence and intracellular survival within human macrophages of Enterococcus faecalis isolates from coastal marine sediment

Enterococcus faecalis is part of the human intestinal microbiota and an important nosocomial pathogen. It can be found in the marine environment, where it is also employed as a fecal indicator. To assess the pathogenic potential of marine E. faecalis, four strains isolated from marine sediment were analyzed for their ability to survive in human macrophages. Escherichia coli DH5α was used as a negative control. The number of adherent and intracellular bacteria was determined 2.5 h after the infection (T₀) and after further 24h (T₂₄) by CFU and qPCR counts. At T₂₄ adherent and intracellular enterococcal CFU counts were increased for all strains, the increment in intracellular bacteria being particularly marked. No CFU of E. coli DH5α were detected. In contrast, qPCR counts of intracellular enterococcal and E. coli bacteria were similar at both time points. These findings suggest that whereas E. coli was killed within macrophages (no CFU, positive qPCR), the E. faecalis isolates not only escaped killing, but actually multiplied, as demonstrated by the increase in the viable cell population. These findings support earlier data by our group, further documenting that marine sediment can be a reservoir of pathogenic enterococci.     

Echinococcus granulosus protoscoleces promotes proliferation and invasion of hepatocellular carcinoma cells

There may exist a connection between Echinococcus granulosus infection and cancer development. Here, it is aimed to investigate specific effects of E. granulosus protoscoleces (PSCs) on the proliferation and invasion capacities of hepatocellular carcinoma (HCC) cells in vitro and ex vitro. HepG2 cells were cultured with different quantities of E. granulosus PSCs in vitro. MTT analysis was used to evaluate effects of E. granulosus PSCs on the proliferation of HepG2 cells. Besides, scratch and transwell assays were respectively used for the detection of HepG2 cells migration and invasion capacities after co-culture with E. granulosus PSCs. Then, HepG2 cells were subcutaneously transplanted into nude mice with or without E. granulosus PSCs. From the 25th day of transplantation, the volume of subcutaneous lesions was measured every four days. At the 37th day, subcutaneous lesions were removed and their weight was evaluated. H&E staining was used for detecting basic pathological changes. HepG2 cells grew well without obvious morphological changes. Proliferation rate and migration capacity of HepG2 cells were higher in the co-culture group than the control group, which was closely associated with quantities of E. granulosus PSCs and co-culture time length. Moreover, HepG2 cells co-cultured with E. granulosus PSCs had stronger invasion ability than the control HepG2 cells. Importantly, there existed significant differences in the volume and weight of subcutaneous lesions after transplanting HepG2 cells with E. granulosus PSCs than the control group. HepG2 cells were also more pathologically heterogeneous in morphology after transplantation with E. granulosus PSCs. Thus, E. granulosus PSCs may promote proliferation and invasion of HCC cells.     

Enhancement of poly(arginyl-histidine) production by Verticillium kibiense E18

An ergot fungus Verticillium kibiense E18 produced two cationic peptides, ɛ-poly-l-lysine (ePL) and poly(l-arginyl-d-histidine) (PRH). The ePL was used as a food preservative, and it was expected that PRH would be used as a novel material, such as cationic and antimicrobial peptide. To enhance PRH production of strain E18, various culture conditions were investigated. Glucose was a suitable carbon source for PRH production, although glycerol was a suitable carbon source for growth. The cultivation temperature significantly influenced both cell growth and PRH production. The optimal temperatures for cell growth and PRH production were 28 and 30 °C, respectively. Moreover, strain E18 produced more PRH when an additional 5.0 μg/L FeSO₄·7H₂O was added to the production medium. Under optimal conditions, strain E18 enhanced PRH production, while suppressing ePL production. The maximum PRH production was 183.9 mg/L, which is approximately 60-fold higher than that of the initial culture condition.     

Development and utilization of new sequenced characterized amplified region markers specific for E genome of Thinopyrum

Species containing E genome of Thinopyrum offered potential to increase the genetic variability and desirable characters for wheat improvement. However, E genome specific marker was rare. The objective of the present report was to develop and identify sequenced characterized amplified region (SCAR) markers that can be used in detecting E chromosome in wheat background for breeding purpose. Total 280 random amplified polymorphic DNA (RAPD) primers were amplified for seeking of E genome specific fragments by using the genomic DNA of Thinopyrum elongatum and wheat controls as templates. As a result, six RAPD fragments specific for E genome were found and cloned, and then were converted to SCAR markers. The usability of these markers was validated using a number of Egenome-containing species and wheat as controls. These markers were subsequently located on E chromosomes using specific PCR and fluorescence in situ hybridization (FISH). SCAR markers developed in this research could be used in molecular marker assisted selection of wheat breeding with Thinopyrum chromatin introgressions.     

Chemical markers of four species of Epimedium used in drug Yin-Yang-Huo

There are four species of Epimedium that have been officially adopted in Chinese Pharmacopoeia (2015) under the same crude drug name ‘Yin-Yang-Huo’, including E. brevicornu Maxim., E. koreanum Nakai., E. sagittatum (Sieb.et Zucc.) and E. pubescens Maxim. This study aimed to identify the chemical markers of these species by HPLC-PDA fingerprinting combined with chemometrics methods. The HPLC separation was performed on a Water^'s Atlantis®T3 column (4.6 × 250 mm, 5 μm) with the gradient elution of acetonitrile-0.1% formic acid aqueous solution using a PDA detector. The HPLC-PDA fingerprints of 88 batches of Yin-Yang-Huo samples were obtained and analysed using principal component analysis (PCA) and hierarchical cluster analysis (HCA). Samples of E. koreanum and E. sagittatum were classified into separate groups, while samples of E. brevicornum and E. pubescens were clustered together. Based on partial least squares-discriminant analysis (PLS-DA) and variable importance in projection, nine potential chemical markers were discovered, and six markers were identified by comparing with reference standards or according to their MS/MS fragmentation behaviour. Among them, korepimedoside C and epimedokoreanoside I could be used as chemical markers of E. koreanum to differentiate it from the other three species, while epimedin B1 and epimedin A1 could be used as chemical markers of E. sagittatum. This study will help effectively distinguish different Yin-Yang-Huo species and provide a valuable strategy for analysing the potential chemical markers of herbal medicines with multiple botanical origins.     

Cloning and Sequencing of the ompA Gene of Enterobacter sakazakii and Development of an ompA-Targeted PCR for Rapid Detection of Enterobacter sakazakii in Infant Formula

Enterobacter sakazakii is an emerging, infant formula-borne pathogen that causes severe meningitis, meningoencephalitis, sepsis, and necrotizing enterocolitis in neonates and infants, with a high fatality rate. Traditional detection methods take up to 7 days to identify E. sakazakii. The outer membrane protein A gene (ompA), along with its flanking sequences from E. sakazakii (ATCC 51329), was cloned in the pGEM-T Easy vector and sequenced. Comparison of the nucleotide and deduced amino acid sequences of the ompA gene with other sequences available in the GenBank database revealed a high degree of homology with ompA genes of other gram-negative bacteria belonging to the Enterobacteriaceae. Based on regions of the ompA gene unique to E. sakazakii, two primers were synthesized to develop and optimize an E. sakazakii-specific PCR. The PCR amplified a 469-bp DNA product from all E. sakazakii strains tested but not from other bacteria. Experiments to determine the sensitivity of the PCR indicated that it could detect as few as 10³ CFU/ml of E. sakazakii bacteria in infant formula directly and 10⁻¹ CFU/ml after an 8-h enrichment step. We conclude that this PCR, combined with enrichment culturing, has the potential to be used as a rapid tool for detecting the presence of E. sakazakii in infant formula.     

De novo synthesis of sphingolipids plays an important role during in vitro encystment of Entamoeba invadens

Entamoeba invadens is a protozoan, which causes multiple damages in reptiles and is considered a prototype for the study of the Entamoeba encystment in vitro. Here we report for the first time the role of the de novo synthesis pathway of sphingolipids during the encystment of E. invadens. In silico analysis showed that this parasite has six putative genes coding for ceramide synthases (CerS), all of them coding for proteins containing the Lag1p motif, a region conserved in the ceramide synthases of multiple organisms, suggesting that they might be bona fide CerS. The six genes of E. invadens are differentially expressed at different time intervals in both stages trophozoite and cyst, based on the results obtained through qRT-PCR assays, the genes involved in the synthesis of sphingolipids with long-chain fatty acids CerS 2,3,4 (EIN_046610, EIN_097030, EIN_130350) have maximum points of relative expression in both stages of the E. invadens life cycle, which strongly suggest that the signaling exerted from the synthesis pathway of sphingolipids is essential for the encystment of E. invadens, since the generation of the more abundant sphingomyelin (SM) subspecies with long-chain fatty acids are fundamental for the parasite to reach its conversion from trophozoite to cyst. When myriocin was used as an inhibitor of serine palmitoyl CoA transferase (SPT), first enzyme in the de novo biosynthesis of sphingolipids, the trophozoites of E. invadens were unable to reach the encystment. Since the effect of myriocin was reversed with exogenous d-erythrosphingosine (DHS), it was demonstrated that the inhibition was specific and it was confirmed that the synthesis of sphingolipids play an essential role during the encystment process of E. invadens.     

A new idea for simple and rapid monitoring of gene expression: requirement of nucleotide sequences encoding an N-terminal HA tag in the T7 promoter-driven expression in E. coli

Mammalian expression vectors are used to overexpress genes of interest in mammalian cells. High temperature requirement protein A1 (HtrA1), used as a specific target, was expressed from the pHA-M-HtrA1 plasmid in HEK293T cells, inducing cell death. Expression of HtrA1 was driven by the pHA-M-HtrA1 mammalian expression vector in E. coli resulting in growth suppression of E. coli in an HtrA1 serine protease-dependent manner. By using various combinations of promoters, target genes and N-terminal tags, the T7 promoter and N-terminal HA tag in the mammalian expression vector were shown to be responsible for expression of target genes in E. coli. Thus the pHA-M-HtrA1 plasmid can be used as a novel, rapid pre-test system for expression and cytotoxicity of the specific target gene in E. coli before assessing its functions in mammalian cells.     

Effect of codon-optimized E. coli signal peptides on recombinant Bacillus stearothermophilus maltogenic amylase periplasmic localization,yield and activity

Recombinant proteins can be targeted to the Escherichia coli periplasm by fusing them to signal peptides. The popular pET vectors facilitate fusion of target proteins to the PelB signal. A systematic comparison of the PelB signal with native E. coli signal peptides for recombinant protein expression and periplasmic localization is not reported. We chose the Bacillus stearothermophilus maltogenic amylase (MA), an industrial enzyme widely used in the baking and brewing industry, as a model protein and analyzed the competence of seven, codon-optimized, E. coli signal sequences to translocate MA to the E. coli periplasm compared to PelB. MA fusions to three of the signals facilitated enhanced periplasmic localization of MA compared to the PelB fusion. Interestingly, these three fusions showed greatly improved MA yields and between 18- and 50-fold improved amylase activities compared to the PelB fusion. Previously, non-optimal codon usage in native E. coli signal peptide sequences has been reported to be important for protein stability and activity. Our results suggest that E. coli signal peptides with optimal codon usage could also be beneficial for heterologous protein secretion to the periplasm. Moreover, such fusions could even enhance activity rather than diminish it. This effect, to our knowledge has not been previously documented. In addition, the seven vector platform reported here could also be used as a screen to identify the best signal peptide partner for other recombinant targets of interest.     

The oligolecty status of a specialist bee of South American Prosopis (Fabaceae) supported by pollen analysis and floral visitation methods

Despite the nest pollen provisions in Western Argentina are composed only of Prosopis, the fact that the ground-nesting bee Eremapis parvula visits several floral hosts suggested that it is a generalist bee species. In the South American Dry Chaco forest, seven nest aggregations of E. parvula were found during three different spring periods. From 34 to 73 species of floral hosts were recorded around each nest aggregation. However, all nest pollen samples were composed of Prosopis pollen alone, as previously found in nests from Western Argentina. Thus, pollen analysis proved that E. parvula is a specialist bee of Prosopis. The “monolecty” and “narrow oligolecty” pollen specialization categories cannot be differentiated using pollen analysis alone. For this reason, a complementary floral visitation method was used. As several Prosopis species have been reported in floral records, the narrow oligolecty category for E. parvula is supported here. Considering that this exomalopsine is a multivoltine bee, and that flowers of some of the more than seven Prosopis species are always available during spring, synchronization between them in rainy periods is highly probable. Thus, it is unlikely that E. parvula has to forage on alternative pollen hosts.     

Identification,Source, and Metabolism of N-Ethylglutamate in Escherichia coli

N-Ethylglutamate (NEG) was detected in Escherichia coli BL21 cells grown on LB broth, and it was found to occur at a concentration of ∼4 mM in these cells under these conditions. The same cells grown on M9 glucose medium contained no detectable amount of NEG. Analysis of the LB broth showed the presence of NEG, a compound never before reported as a natural product. Isotope dilution analysis showed that it occurred at a concentration of 160 μM in LB broth. Analyses of yeast extract and tryptone, the organic components of LB broth, both showed the presence NEG. It was demonstrated that NEG can be generated during the autolysis of the yeast used in the preparation of the yeast extract. Growth of these E. coli cells in LB broth prepared in deuterated water showed no incorporation of deuterium into NEG, demonstrating that E. coli cells did not generate the NEG. Cell growth rates were not affected by the addition of 5 mM NEG to either LB or M9 glucose medium. l-[ethyl-²H₄]NEG was found to be readily incorporated into the cells and metabolized by the cells. From these results, it was concluded that all of the NEG present in the cells was taken up from the medium. NEG could serve as the sole nitrogen source for E. coli when grown on M9 glucose medium in the presence of glucose but could not serve as the sole carbon source on M9 medium in the absence of glucose.During work on developing methods for the analysis of the amino acids generated by recombinant archaeal mutases, I developed procedures for the recovery and analysis of the free amino acids present in cell extracts of Escherichia coli. When these methods were applied to analysis of E. coli grown on LB broth, I always found a large amount of an unknown amino acid. Here I report on the identification of this amino acid as N-ethylglutamate (NEG). NEG has never been reported as a natural product. I demonstrate that NEG is readily taken up by E. coli and can serve as the sole source of nitrogen when the cells are grown on M9 glucose medium.     

Enhanced Heterologous Expression of Two Streptomyces griseolus Cytochrome P450s and Streptomyces coelicolor Ferredoxin Reductase as Potentially Efficient Hydroxylation Catalysts

The herbicide-inducible, soluble cytochrome P450s CYP105A1 and CYP105B1 and their adjacent ferredoxins, Fd1 and Fd2, of Streptomyces griseolus were expressed in Escherichia coli to high levels. Conditions for high-level expression of active enzyme able to catalyze hydroxylation have been developed. Analysis of the expression levels of the P450 proteins in several different E. coli expression hosts identified E. coli BL21 Star(DE3)pLysS as the optimal host cell to express CYP105B1 as judged by CO difference spectra. Examination of the codons used in the CYP1051A1 sequence indicated that it contains a number of codons corresponding to rare E. coli tRNA species. The level of its expression was improved in the modified forms of E. coli BL21(DE3), which contain extra copies of rare codon E. coli tRNA genes. The activity of correctly folded cytochrome P450s was further enhanced by cloning a ferredoxin reductase from Streptomyces coelicolor downstream of CYP105A1 and CYP105B1 and their adjacent ferredoxins. Expression of CYP105A1 and CYP105B1 was also achieved in Streptomyces lividans 1326 by cloning the P450 genes and their ferredoxins into the expression vector pBW160. S. lividans 1326 cells containing CYP105A1 or CYP105B1 were able efficiently to dealkylate 7-ethoxycoumarin.     

Entamoeba histolytica and Entamoeba invadens: Effects of temperature and oxygen tension on growth and survival

The temperature ranges for axenic growth of Entamoeba histolytica strain HM-1 and Entamoeba invadens strain 165 clone II in TYI-S-33 medium were: 32 to 40 C for E. histolytica with an optimum of 35.5 to 37 C; whereas the range for E. invadens 165 II was 20 to 30 C, optimum 24 to 27 C. Growth of strain HM-1 at 40 C was dependent upon the cell density of the culture used as a source of the inoculum. Clonal growth in agar was used to assay survival of Entamoeba spp. trophozoites after exposure to deleterious physical conditions. The lowest temperature for thermal killing of E. histolytica HM-1 was 41.5 C and for E. invadens 165, 35.5 C. Both were killed rapidly at 42 C, but slowly at 0 C. Killing of E. histolytica HM-1 trophozoites by exposure to increased oxygen tensions was a function of temperature and time. At 24 C and 35.5 C, there was little loss of viability during the first 7 hr exposure, then killing was quite rapid. The cells were killed sooner if the medium was preexposed to air. In contrast, at 0 C there was less killing by oxygen. E. invadens 165 II appeared to be more oxygen sensitive at 24 than at 0 C. Other E. histolytica strains tested were similar to HM-1 in their responses to temperature and air.