High-Throughput Profiling of Caenorhabditis elegans Starvation-Responsive microRNAs

MicroRNAs (miRNAs) are non-coding RNAs of ~22 nucleotides in length that regulate gene expression by interfering with the stability and translation of mRNAs. Their expression is regulated during development, under a wide variety of stress conditions and in several pathological processes. In nature, animals often face feast or famine conditions. We observed that subjecting early L4 larvae from Caenorhabditis elegans to a 12-hr starvation period produced worms that are thinner and shorter than well-fed animals, with a decreased lipid accumulation, diminished progeny, reduced gonad size, and an increased lifespan. Our objective was to identify which of the 302 known miRNAs of C. elegans changed their expression under starvation conditions as compared to well-fed worms by means of deep sequencing in early L4 larvae. Our results indicate that 13 miRNAs (miR-34-3p, the family of miR-35-3p to miR-41-3p, miR-39-5p, miR-41-5p, miR-240-5p, miR-246-3p and miR-4813-5p) were upregulated, while 2 miRNAs (let-7-3p and miR-85-5p) were downregulated in 12-hr starved vs. well-fed early L4 larvae. Some of the predicted targets of the miRNAs that changed their expression in starvation conditions are involved in metabolic or developmental process. In particular, miRNAs of the miR-35 family were upregulated 6–20 fold upon starvation. Additionally, we showed that the expression of gld-1, important in oogenesis, a validated target of miR-35-3p, was downregulated when the expression of miR-35-3p was upregulated. The expression of another reported target, the cell cycle regulator lin-23, was unchanged during starvation. This study represents a starting point for a more comprehensive understanding of the role of miRNAs during starvation in C. elegans.     

Survival rates of mutant genes under artificial selection using individual and family information

We have used diffusion and branching process methods to investigate fixation rates, probabilities of survival per generation, and times to fixation of mutant genes under different selection methods incorporating individual and family information. Diffusion approximations fit well to simulated results even for large selection coefficients. Methods that give much weight to family information, such as BLUP evaluation which is widely used in animal breeding, reduce fixation rates of mutant genes because of the reduced effective population sizes. In general, it is observed that even mutants with relatively small heterozygous effects (say 0.1 phenotypic standard deviation) are practically ‘safe’ (i.e. their probability of loss from one generation to the next is smaller than, say, 10%) after just a few generations, typically less than 10. For methods of selection with larger effective size, such as within-family selection, the mutant is ‘safe’ in the population somewhat earlier but eventual fixation takes a longer time. Finally we evaluate the amount by which the use of marker assisted selection reduces the fixation probability of newly arisen mutants.     

Pore-forming Activity of the Escherichia coli Type III Secretion System Protein EspD

Enterohemorrhagic Escherichia coli is a causative agent of gastrointestinal and diarrheal diseases. Pathogenesis associated with enterohemorrhagic E. coli involves direct delivery of virulence factors from the bacteria into epithelial cell cytosol via a syringe-like organelle known as the type III secretion system. The type III secretion system protein EspD is a critical factor required for formation of a translocation pore on the host cell membrane. Here, we show that recombinant EspD spontaneously integrates into large unilamellar vesicle (LUV) lipid bilayers; however, pore formation required incorporation of anionic phospholipids such as phosphatidylserine and an acidic pH. Leakage assays performed with fluorescent dextrans confirmed that EspD formed a structure with an inner diameter of ∼2.5 nm. Protease mapping indicated that the two transmembrane helical hairpin of EspD penetrated the lipid layer positioning the N- and C-terminal domains on the extralumenal surface of LUVs. Finally, a combination of glutaraldehyde cross-linking and rate zonal centrifugation suggested that EspD in LUV membranes forms an ∼280–320-kDa oligomeric structure consisting of ∼6–7 subunits.     

The Role of Protein Denaturation Energetics and Molecular Chaperones in the Aggregation and Mistargeting of Mutants Causing Primary Hyperoxaluria Type I

Primary hyperoxaluria type I (PH1) is a conformational disease which result in the loss of alanine:glyoxylate aminotransferase (AGT) function. The study of AGT has important implications for protein folding and trafficking because PH1 mutants may cause protein aggregation and mitochondrial mistargeting. We herein describe a multidisciplinary study aimed to understand the molecular basis of protein aggregation and mistargeting in PH1 by studying twelve AGT variants. Expression studies in cell cultures reveal strong protein folding defects in PH1 causing mutants leading to enhanced aggregation, and in two cases, mitochondrial mistargeting. Immunoprecipitation studies in a cell-free system reveal that most mutants enhance the interactions with Hsc70 chaperones along their folding process, while in vitro binding experiments show no changes in the interaction of folded AGT dimers with the peroxisomal receptor Pex5p. Thermal denaturation studies by calorimetry support that PH1 causing mutants often kinetically destabilize the folded apo-protein through significant changes in the denaturation free energy barrier, whereas coenzyme binding overcomes this destabilization. Modeling of the mutations on a 1.9 Å crystal structure suggests that PH1 causing mutants perturb locally the native structure. Our work support that a misbalance between denaturation energetics and interactions with chaperones underlie aggregation and mistargeting in PH1, suggesting that native state stabilizers and protein homeostasis modulators are potential drugs to restore the complex and delicate balance of AGT protein homeostasis in PH1.     

S-adenosylmethionine decarboxylase from Leishmania donovani. Molecular, genetic, and biochemical characterization of null mutants and overproducers.

The polyamine biosynthetic enzyme, S-adenosylmethionine decarboxylase (ADOMETDC) has been advanced as a potential target for antiparasitic chemotherapy. To investigate the importance of this protein in a model parasite, the gene encoding ADOMETDC has been cloned and sequenced from Leishmania donovani. The Delta adometdc null mutants were created in the insect vector form of the parasite by double targeted gene replacement. The Delta adometdc strains were incapable of growth in medium without polyamines; however, auxotrophy could be rescued by spermidine but not by putrescine, spermine, or methylthioadenosine. Incubation of Delta adometdc parasites in medium lacking polyamines resulted in a drastic increase of putrescine and glutathione levels with a concomitant decrease in the amounts of spermidine and the spermidine-containing thiol trypanothione. Parasites transfected with an episomal ADOMETDC construct were created in both wild type and Delta adometdc parasites. ADOMETDC overexpression abrogated polyamine auxotrophy in the Delta adometdc L. donovani. In addition, ADOMETDC overproduction in wild type parasites alleviated the toxic effects of 5'-(((Z)-4-amino-2-butenyl)methylamino)-5'-deoxyadenosine (MDL 73811), but not pentamidine, berenil, or methylglyoxyl bis(guanylhydrazone), all inhibitors of ADOMETDC activities in vitro. The molecular, biochemical, and genetic characterization of ADOMETDC establishes that it is essential in L. donovani promastigotes and a potential target for therapeutic validation.     

A synoptical revision of Blepharidachne (Poaceae)

Blepharidachne is a disjunct American genus with four species:B. kingii (S. Wats.) Hackel (Great Basin of western U.S.A.) andB. bigelovii (S. Wats.) Hackel (Coahuila, Mexico and Texas, U.S.A.) in North America;B. benthamiana (Hackel) A. S. Hitchc. andB. hitchcockii Lahitte, both in central and western Argentina.Blepharidachne kingii is the only species with perfect flowers, the other three being monoecious as a result of a series of reductive processes. After a discussion of these morphological peculiarities, heretofore overlooked, the article is completed with a brief taxonomic synopsis of the genus.