Construction of transgenic mice with Δ15 Des enzyme activity by using a PiggyBac transposon / 生物工程学报

Essential fatty acids are those that could not be synthesized by the body itself but crucial for health and life. Studies have shown that ω-3 fatty acids may facilitate human physiological functions. Mammals lack ω-3 desaturase gene, and the Δ15 fatty acid desaturase (Δ15 Des) from Caenorhabditis elegans can transform the ω-6 polyunsaturated fatty acids (PUFAs) into ω-3 PUFAs. Transgenic mice expressing Δ15 Des enzyme activity was constructed by using a PiggyBac transposon (PB). Homozygous transgenic mice with stable inheritance was bred in a short time, with a positive rate of 35.1% achieved. The mice were fed with 6% ω-6 PUFAs and the changes of fatty acids in mice were detected by gas chromatography (GC). The expression level of Δ15 Des in mice was detected by quantitative PCR (qPCR) and Western blotting (WB). qPCR and GC analysis revealed that the percentage of positive mice harboring the active gene was 61.53%. Compared with traditional methods, the transformation efficiency and activity of Δ15 Des were significantly improved, and homozygotes showed higher activity than that of heterozygotes. This further verified the efficient transduction efficiency of the PiggyBac transposon system.

Isolamento e caraterização de Lactobacillus spp. da cavidade bucal e sua ação probiótica sob Candida albicans: formação de biofilme, infecção em modelos de invertebrados e expressão dos genes EFG1, HWP1 e ALS1 / Isolation and characterization of Lactobacillus spp. from oral cavity and their probiotic action in Candida albicans: biofilm formation, infection in invertebrate models and EFG1, HWP1 and ALS1 gene expression

O estudo da atividade inibitória de Lactobacillus pode contribuir na descoberta de novas estratégias terapêuticas nas infecções por Candida. Nesse contexto, o objetivo desse estudo foi isolar e identificar Lactobacillus da cavidade bucal de indivíduos livres de cárie e avaliar seu potencial de inibição de C. albicans por meio de estudos in vitro e in vivo. Primeiramente, foram avaliados os efeitos de 30 isolados clínicos de Lactobacillus sobre o número de células viáveis (UFC) em biofilme de C. albicans e sobre a formação de hifas. Os isolados que obtiveram os maiores efeitos inibitórios sobre C. albicans foram selecionados para os testes de determinação da biomassa total dos biofilmes pela absorbância do cristal violeta, análise da arquitetura dos biofilmes por microscopia eletrônica de varredura (MEV) e quantificação da expressão de genes de C. albicans (ALS3, HWP1, EFG1 e CPH1) por qPCR. Esses isolados também foram submetidos a estudos in vivo usando os modelos de Galleria mellonella e Caenorhabditis elegans. Para o estudo em G. mellonella, a infecção experimental foi avaliada pela curva de sobrevivência, quantificação da carga fúngica na hemolinfa, densidade hemocitária, quantificação da expressão gênica de peptídeos antifúngicos (Gallerymicina e Galiomicina) e monitoramento da infecção de C. albicans por análise de bioluminescência. No modelo de C. elegans, a infecção foi avaliada por meio dos ensaios de curva de sobrevivência e estudo da filamentação de C. albicans. Os resultados dos ensaios in vitro demonstraram que L. paracasei 28.4, L. rhamnosus 5.2 e L. fermentum 20.4 foram as cepas com maior atividade antimicrobiana sobre os biofilmes de C. albicans. Nessas cepas, todos os genes analisados foram regulados negativamente na associação com Lactobacillus quando comparados com o grupo controle. No estudo in vivo, a injeção de L. paracasei 28.4 em G. mellonella infectadas com C. albicans aumentou a sobrevida das larvas, o número de hemócitos e a expressão de peptídeos antifúngicos, reduzindo assim a UFC de C. albicans. Em C. elegans, L. paracasei 28.4 também foi capaz de aumentar a sobrevida dos vermes infectados com C. albicans e reduzir a filamentação. Conclui-se que L. fermentum 20.4, L. paracasei 28.4 e L. rhamnosus 5.2 tem potencial para serem usados como probióticos na cavidade bucal devido sua ação anti-biofilme e sua regulação negativa dos genes de virulência de C. albicans. L. paracasei 28.4 foi capaz de prolongar a sobrevida nos dois modelos experimentais infectados com C. albicans por apresentarem ação antifúngica e imunomodulatória(AU)

The study of the antifungal activity of Lactobacillus may contribute to the discovery of new therapeutic strategies for Candida infections. In this context, the objective of this study was to isolate and identify Lactobacillus from the oral cavity of caries-free subjects and to evaluate its effects through in vitro and in vivo studies. First, the effects of 30 clinical isolates of Lactobacillus on the number of viable cells (CFU) in biofilms of C. albicans and on hyphae formation were evaluated. The isolates that obtained the highest inhibitory effects on C. albicans were selected for biofilm biomass determination by violet crystal absorbance, analysis of biofilm architecture by scanning electron microscopy (SEM) and quantification of the expression of C. albicans (ALS3, HWP1, EFG1 and CPH1) by real time PCR. These isolates were also submitted to in vivo studies using the Galleria mellonella and Caenorhabditis elegans models. For the study in the model of Galleria mellonella, the experimental infection was evaluated by the survival curve, quantification of the fungal load in the hemolymph, hemocitary density, the gene expression of antifungal peptides (Gallerymicin and Galiomicin) and monitoring of C. albicans infection by bioluminescence analysis. In the Caenorhabditis elegans model, the infection was evaluated by the survival curve assays and the study of C. albicans filamentation. The results of in vitro tests demonstrated that L. paracasei 28.4, L. rhamnosus 5.2 and L. fermentum 20.4 were the strains with the highest antimicrobial activity on the biofilms of C. albicans. In these strains, all analyzed genes were negatively regulated in association with Lactobacillus when compared to the control group. In the in vivo study, the injection of L. paracasei 28.4 into the G. mellonella increased survival of the larvae, the number of hemocytes and the expression of antifungal peptides, thus reducing the CFU of C. albicans. In C. elegans, L. paracasei 28.4 was also able to increase the survival of worms infected with C. albicans and reduce the filamentation. We conclude that L. fermentum 20.4, L. paracasei 28.4 and L. rhamnosus 5.2 have potential to be used as probiotics in the oral cavity due to their anti-biofilm action and their negative regulation of virulence genes of C. albicans. L. paracasei 28.4 was able to prolong survival of both experimental models infected with C. albicans for having antifungal and immunomodulatory action(AU)

Electrolyzed-reduced water increases resistance to oxidative stress, fertility, and lifespan via insulin/IGF-1-like signal in C. elegans

Electrolyzed-reduced water (ERW) scavenges reactive oxygen species and is a powerful anti-oxidant. A positive correlation between oxidative stress and aging has been proved in many model organisms. In Caenorhabditis elegans, many long-lived mutants showed reduced fertility as a trade off against longevity phenotype. We aimed to study the effect of ERW on oxidative stress, fertility and lifespan of C. elegans. We also investigated the genetic pathway involved in the effect of ERW on resistance to oxidative stress and lifespan. We compared lifespan and fertility of worms in media prepared with distilled water and ERW. ERW significantly extended lifespan and increased the number of progeny produced. Then the effect of ERW on resistance to oxidative stress and lifespan of long-lived mutants was determined. ERW increased resistance to oxidative stress and lifespan of eat-2, a genetic model of dietary restriction, but had no effect on those of age-1, which is involved in insulin/insulin-like growth factor (IGF)-1-like signal. In addition, knockdown of daf-16, the downstream mediator of insulin/IGF-1-like signal, completely prevented the effect of ERW on lifespan. These findings suggest that ERW can extend lifespan without accompanying reduced fertility and modulate resistance to oxidative stress and lifespan via insulin/IGF-1-like signal in C. elegans.

Diet-dependent depletion of queuosine in tRNAs in Caenorhabditis elegans does not lead to a developmental block.

Queuosine (Q), a hypermodified nucleoside,occurs at the wobble position of transfer RNAs (tRNAs)with GUN anticodons. In eubacteria, absence of Q affects messenger RNA (mRNA) translation and reduces the virulence of certain pathogenic strains. In animal cells,changes in the abundance of Q have been shown to correlate with diverse phenomena including stress tolerance, cell proliferation and tumour growth but the function of Q in animals is poorly understood. Animals are thought to obtain Q (or its analogues) as a micronutrient from dietary sources such as gut micro flora. However,the difficulty of maintaining animals under bacteria-free conditions on Q-deficient diets has severely hampered the study of Q metabolism and function in animals. In this study,we show that as in higher animals, tRNAs in the nematode Caenorhabditis elegans are modified by Q and its sugar derivatives. When the worms were fed on Q-deficient Escherichia coli, Q modification was absent from the worm tRNAs suggesting that C.elegans lacks a de novo pathway of Q biosynthesis. The inherent advantages of C.elegans as a model organism, and the simplicity of conferring a Q-deficient phenotype on it make it an ideal system to investigate the function of Q modification in tRNA.

The RNA interference revolution

The discovery of double-stranded RNA-mediated gene silencing has rapidly led to its use as a method of choice for blocking a gene, and has turned it into one of the most discussed topics in cell biology. Although still in its infancy, the field of RNA interference has already produced a vast array of results, mainly in Caenorhabditis elegans, but recently also in mammalian systems. Micro-RNAs are short hairpins of RNA capable of blocking translation, which are transcribed from genomic DNA and are implicated in several aspects from development to cell signaling. The present review discusses the main methods used for gene silencing in cell culture and animal models, including the selection of target sequences, delivery methods and strategies for a successful silencing. Expected developments are briefly discussed, ranging from reverse genetics to therapeutics. Thus, the development of the new paradigm of RNA-mediated gene silencing has produced two important advances: knowledge of a basic cellular mechanism present in the majority of eukaryotic cells and access to a potent and specific new method for gene silencing.

Accelerated molecular evolution of insect orthologues of ERG28/C14orf1: a link with ecdysteroid metabolism?

We have analysed the evolution of ERG28/C14orf1, a gene coding for a protein involved in sterol biosynthesis. While primary sequence of the protein is well conserved in all organisms able to synthesize sterols de novo, strong divergence is noticed in insects, which are cholesterol auxotrophs. In spite of this virtual acceleration, our analysis suggests that the insect orthologues are evolving today at rates similar to those of the remaining members of the family. A plausible way to explain this acceleration and subsequent stabilization is that Erg28 plays a role in at least two different pathways. Discontinuation of the cholesterogenesis pathway in insects allowed the protein to evolve as much as the function in the other pathway was not compromised.

Ageing, gerontogenes, and hormesis.

Evolutionary theories of ageing and longevity argue against the existence of specific genes that cause ageing. However, genes whose altered activity influences ageing and longevity, may be termed gerontogenes. Several putative gerontogenes have been identified in various ageing systems, including the Drosophila, budding yeast, nematodes and cells in culture. Since ageing is characterized by a progressive failure of maintenance and repair, it is reasoned that genes involved in homeodynamic repair pathways are the most likely candidate gerontogenes. A promising approach for the identification of critical gerontogenic processes is hormesis-like positive effects of stress. Stimulation of various repair pathways by mild stress has significant effects on delaying the onset of various age-associated alterations in cells, tissues and organisms.