Characterization of yeast strains with conditionally expressed variants of ribosomal protein genes tcm1 and cyh2.

We placed a regulatory sequence derived from the GAL10 locus of Saccharomyces cerevisiae at various distances from the start sites of transcription of two yeast ribosomal protein genes, tcm1 and cyh2. The hybrid ribosomal protein genes were transcribed at wild-type levels in the presence of galactose. In the absence of galactose, the hybrid genes were transcribed either at a reduced level or essentially not at all. Yeast cells which transcribe the ribosomal protein genes at a reduced rate continued to grow, suggesting that enhanced translation of the ribosomal protein mRNA may permit an adequate rate of synthesis of the corresponding protein. Consistent with this suggestion is the finding that preexisting mRNA decayed at a reduced rate when transcription was halted abruptly by removal of galactose. Yeast cells unable to transcribe tcm1 or cyh2 without galactose did not grow. These conditional lethal strains demonstrate that the ribosomal proteins encoded by tcm1 and cyh2 are essential; furthermore, these strains are potentially useful for isolating mutations in the tcm1 and cyh2 proteins affecting their transport, assembly, or function

Physiological concentrations of bile acids down-regulate agonist induced secretion in colonic epithelial cells

In patients with bile acid malabsorption, high concentrations of bile acids enter the colon and stimulate Cl− and fluid secretion, thereby causing diarrhoea. However, deoxycholic acid (DCA), the predominant colonic bile acid, is normally present at lower concentrations where its role in regulating transport is unclear. Thus, the current study set out to investigate the effects of physiologically relevant DCA concentrations on colonic epithelial secretory function. Cl− secretion was measured as changes in short-circuit current across voltage-clamped T84 cell monolayers. At high concentrations (0.5–1 mM), DCA acutely stimulated Cl− secretion but this effect was associated with cell injury, as evidenced by decreased transepithelial resistance (TER) and increased lactate dehydrogenase (LDH) release. In contrast, chronic (24 hrs) exposure to lower DCA concentrations (10–200 μM) inhibited responses to Ca2+ and cAMP-dependent secretagogues without altering TER, LDH release, or secretagogue-induced increases in intracellular second messengers. Other bile acids – taurodeoxycholic acid, chenodeoxycholic acid and cholic acid – had similar antisecretory effects. DCA (50 μM) rapidly stimulated phosphorylation of the epidermal growth factor receptor (EGFr) and both ERK and p38 MAPKs (mitogen-activated protein kinases). The EGFr inhibitor, AG1478, and the protein synthesis inhibitor, cycloheximide, reversed the antisecretory effects of DCA, while the MAPK inhibitors, PD98059 and SB203580, did not. In summary, our studies suggest that, in contrast to its acute prosecretory effects at pathophysiological concentrations, lower, physiologically relevant, levels of DCA chronically down-regulate colonic epithelial secretory function. On the basis of these data, we propose a novel role for bile acids as physiological regulators of colonic secretory capacity

Diminished Self-Chaperoning Activity of the ΔF508 Mutant of CFTR Results in Protein Misfolding

The absence of a functional ATP Binding Cassette (ABC) protein called the Cystic Fibrosis Transmembrane Conductance Regulator (CFTR) from apical membranes of epithelial cells is responsible for cystic fibrosis (CF). Over 90% of CF patients carry at least one mutant allele with deletion of phenylalanine at position 508 located in the N-terminal nucleotide binding domain (NBD1). Biochemical and cell biological studies show that the ΔF508 mutant exhibits inefficient biosynthetic maturation and susceptibility to degradation probably due to misfolding of NBD1 and the resultant misassembly of other domains. However, little is known about the direct effect of the Phe508 deletion on the NBD1 folding, which is essential for rational design strategies of cystic fibrosis treatment. Here we show that the deletion of Phe508 alters the folding dynamics and kinetics of NBD1, thus possibly affecting the assembly of the complete CFTR. Using molecular dynamics simulations, we find that meta-stable intermediate states appearing on wild type and mutant folding pathways are populated differently and that their kinetic accessibilities are distinct. The structural basis of the increased misfolding propensity of the ΔF508 NBD1 mutant is the perturbation of interactions in residue pairs Q493/P574 and F575/F578 found in loop S7-H6. As a proof-of-principle that the S7-H6 loop conformation can modulate the folding kinetics of NBD1, we virtually design rescue mutations in the identified critical interactions to force the S7-H6 loop into the wild type conformation. Two redesigned NBD1-ΔF508 variants exhibited significantly higher folding probabilities than the original NBD1-ΔF508, thereby partially rescuing folding ability of the NBD1-ΔF508 mutant. We propose that these observed defects in folding kinetics of mutant NBD1 may also be modulated by structures separate from the 508 site. The identified structural determinants of increased misfolding propensity of NBD1-ΔF508 are essential information in correcting this pathogenic mutant

Release of Lungworm Larvae from Snails in the Environment: Potential for Alternative Transmission Pathways

Background: Gastropod-borne parasites may cause debilitating clinical conditions in animals and humans following the consumption of infected intermediate or paratenic hosts. However, the ingestion of fresh vegetables contaminated by snail mucus and/or water has also been proposed as a source of the infection for some zoonotic metastrongyloids (e.g., Angiostrongylus cantonensis). In the meantime, the feline lungworms Aelurostrongylus abstrusus and Troglostrongylus brevior are increasingly spreading among cat populations, along with their gastropod intermediate hosts. The aim of this study was to assess the potential of alternative transmission pathways for A. abstrusus and T. brevior L3 via the mucus of infected Helix aspersa snails and the water where gastropods died. In addition, the histological examination of snail specimens provided information on the larval localization and inflammatory reactions in the intermediate host. Methodology/Principal Findings: Twenty-four specimens of H. aspersa received ~500 L1 of A. abstrusus and T. brevior, and were assigned to six study groups. Snails were subjected to different mechanical and chemical stimuli throughout 20 days in order to elicit the production of mucus. At the end of the study, gastropods were submerged in tap water and the sediment was observed for lungworm larvae for three consecutive days. Finally, snails were artificially digested and recovered larvae were counted and morphologically and molecularly identified. The anatomical localization of A. abstrusus and T. brevior larvae within snail tissues was investigated by histology. L3 were detected in the snail mucus (i.e., 37 A. abstrusus and 19 T. brevior) and in the sediment of submerged specimens (172 A. abstrusus and 39 T. brevior). Following the artificial digestion of H. aspersa snails, a mean number of 127.8 A. abstrusus and 60.3 T. brevior larvae were recovered. The number of snail sections positive for A. abstrusus was higher than those for T. brevior. Conclusions: Results of this study indicate that A. abstrusus and T. brevior infective L3 are shed in the mucus of H. aspersa or in water where infected gastropods had died submerged. Both elimination pathways may represent alternative route(s) of environmental contamination and source of the infection for these nematodes under field conditions and may significantly affect the epidemiology of feline lungworms. Considering that snails may act as intermediate hosts for other metastrongyloid species, the environmental contamination by mucus-released larvae is discussed in a broader context

Genetic Biocontrol for Invasive Species

Invasive species are increasingly affecting agriculture, food, fisheries, and forestry resources throughout the world. As a result of global trade, invasive species are often introduced into new environments where they become established and cause harm to human health, agriculture, and the environment. Prevention of new introductions is a high priority for addressing the harm caused by invasive species, but unfortunately efforts to prevent new introductions do not address the economic harm that is presently manifested where invasive species have already become established. Genetic biocontrol can be defined as the release of organisms with genetic methods designed to disrupt the reproduction of invasive populations. While these methods offer the potential to control or even eradicate invasive species, there is a need to ensure that genetic biocontrol methods can be deployed in a way that minimizes potential harm to the environment. This review provides an overview of the state of genetic biocontrol, focusing on several approaches that were the subject of presentations at the Genetic Biocontrol for Invasive Species Workshop in Tarragona, Spain, March 31st, 2019, a workshop sponsored by the OECD’s Co-operative Research Program on Biological Resource Management for Sustainable Agricultural Systems. The review considers four different approaches to genetic biocontrol for invasive species; sterile-release, YY Males, Trojan Female Technique, and gene drive. The different approaches will be compared with respect to the efficiency each affords as a genetic biocontrol tool, the practical utility and cost/benefits associated with implementation of the approach, and the regulatory considerations that will need to be addressed for each. The opinions expressed and arguments employed in this publication are the sole responsibility of the authors and do not necessarily reflect those of the OECD or of the governments of its Member countries

A BBP–Mud2p heterodimer mediates branchpoint recognition and influences splicing substrate abundance in budding yeast

The 3′ end of mammalian introns is marked by the branchpoint binding protein, SF1, and the U2AF65-U2AF35 heterodimer bound at an adjacent sequence. Baker's yeast has equivalent proteins, branchpoint binding protein (BBP) (SF1) and Mud2p (U2AF65), but lacks an obvious U2AF35 homolog, leaving open the question of whether another protein substitutes during spliceosome assembly. Gel filtration, affinity selection and mass spectrometry were used to show that rather than a U2AF65/U2AF35-like heterodimer, Mud2p forms a complex with BBP without a third (U2AF35-like) factor. Using mutants of MUD2 and BBP, we show that the BBP–Mud2p complex bridges partner-specific Prp39p, Mer1p, Clf1p and Smy2p two-hybrid interactions. In addition to inhibiting Mud2p association, the bbpΔ56 mutation impairs splicing, enhances pre-mRNA release from the nucleus, and similar to a mud2::KAN knockout, suppresses a lethal sub2::KAN mutation. Unexpectedly, rather than exacerbating bbpΔ56, the mud2::KAN mutation partially suppresses a pre-mRNA accumulation defect observed with bbpΔ56. We propose that a BBP–Mud2p heterodimer binds as a unit to the branchpoint in vivo and serves as a target for the Sub2p-DExD/H-box ATPase and for other splicing factors during spliceosome assembly. In addition, our results suggest the possibility that the Mud2p may enhance the turnover of pre-mRNA with impaired BBP-branchpoint association

Are p.I148T, p.R74W and p.D1270N cystic fibrosis causing mutations ?

BACKGROUND: To contribute further to the classification of three CFTR amino acid changes (p.I148T, p.R74W and p.D1270N) either as CF or CBAVD-causing mutations or as neutral variations. METHODS: The CFTR genes from individuals who carried at least one of these changes were extensively scanned by a well established DGGE assay followed by direct sequencing and familial segregation analysis of mutations and polymorphisms. RESULTS: Four CF patients (out of 1238) originally identified as carrying the p.I148T mutation in trans with a CF mutation had a second mutation (c.3199del6 or a novel mutation c.3395insA) on the p.I148T allele. We demonstrate here that the deletion c.3199del6 can also be associated with CF without p.I148T. Three CBAVD patients originally identified with the complex allele p.R74W-p.D1270N were also carrying p.V201M on this allele, by contrast with non CF or asymptomatic individuals including the mother of a CF child, who were carrying p.R74W-p.D1270N alone. CONCLUSION: These findings question p.I148T or p.R74W-p.D1270N as causing by themselves CF or CBAVD and emphazises the necessity to perform a complete scanning of CFTR genes and to assign the parental alleles when novel missense mutations are identified