Sows affect their piglets’ faecal microbiota until fattening but not their Salmonella enterica shedding status

Recent studies have shown that Salmonella shedding status affects sows’ microbiota during gestation and that these modifications are reflected in the faecal microbiota of their piglets at weaning. The aims of this study were: (a) to evaluate the persistence, up to the fattening period, of the previously measured link between the microbiota of piglets and their mothers’ Salmonella shedding status; and (b) measure the impact of the measured microbiota variations on their Salmonella excretion at this stage. To achieve this, 76 piglets born from 19 sows for which the faecal microbiota was previously documented, were selected in a multisite production system. The faecal matter of these swine was sampled after 4 weeks, at the fattening stage. The Salmonella shedding status and faecal microbiota of these animals were described using bacteriological and 16S rRNA gene amplicon sequencing respectively. The piglet digestive microbiota association with the Salmonella shedding status of their sows did not persist after weaning and did not affect the risk of Salmonella excretion during fattening, while the birth mother still affected the microbiota of the swine at fattening. This supports the interest in sows as a target for potentially transferrable microbiota modifications.     

Crystal structure of a novel mid-gut procarboxypeptidase from the cotton pest Helicoverpa armigera.

The cotton bollworm Helicoverpa armigera (Hubner) (Lepidoptera: Noctuidae) is one of the most serious insect pests in Australia, India and China. The larva causes substantial economical losses to legume, fibre, cereal oilseed and vegetable crops. This pest has proven to be difficult to control by conventional means, mainly due to the development of pesticide resistance. We present here the 2.5 A crystal structure from the novel procarboxypeptidase (PCPAHa) found in the gut extracts from H. armigera larvae, the first one reported for an insect. This metalloprotease is synthesized as a zymogen of 46.6 kDa which, upon in vitro activation with Lys-C endoproteinase, yields a pro-segment of 91 residues and an active carboxypeptidase moiety of 318 residues. Both regions show a three-dimensional structure quite similar to the corresponding structures in mammalian digestive carboxypeptidases, the most relevant structural differences being located in the loops between conserved secondary structure elements, including the primary activation site. This activation site contains the motif (Ala)(5)Lys at the C terminus of the helix connecting the pro- and the carboxypeptidase domains. A remarkable feature of PCPAHa is the occurrence of the same (Ala)(6)Lys near the C terminus of the active enzyme. The presence of Ser255 in PCPAHa instead of Ile and Asp found in the pancreatic A and B forms, respectively, enlarges the S1' specificity pocket and influences the substrate preferences of the enzyme. The C-terminal tail of the leech carboxypeptidase inhibitor has been modelled into the PCPAHa active site to explore the substrate preferences and the enzymatic mechanism of this enzyme.     

Repetitive sequences of the sea urchin genome: Distribution of members of specific repetitive families

Three repetitive sequence families from the sea urchin genome were studied, each defined by homology with a specific cloned probe one to a few hundred nucleotides long. Recombinant λ-sea urchin DNA libraries were screened with these probes, and individual recombinants were selected that include genomic members of these families. Restriction mapping, gel blot, and kinetic analyses were carried out to determine the organization of each repeat family. Sequence elements belonging to the first of the three repeat families were found to be embedded in longer repeat sequences. These repeat sequences frequently occur in small clusters. Members of the second repeat family are also found in a long repetitive sequence environment, but these repeats usually occur singly in any given region of the DNA. The sequences of the third repeat are only 200 to 300 nucleotides long, and are generally terminated by single copy DNA, though a few examples were found associated with other repeats. These three repeat sequence families constitute sets of homologous sequence elements that relate distant regions of the DNA.     

Intensive RNAi with lentiviral vectors in mammalian cells

RNAi is a powerful technology for analyzing gene function in human cells. However, its utility can be compromised by inadequate knockdown of the target mRNA or by interpretation of effects without rigorous controls. We review lentiviral vector-based methods that enable transient or stable knockdowns to trace mRNA levels in human CD4+ T cell lines and other targets. Critical controls are reviewed, including rescue of the pre-knockdown phenotype by re-expression of the targeted gene. The time from thinking about a potential knockdown target to analysis of phenotypes can be as short as a few weeks.     

The ubiquitin C‐terminal hydrolase UCH‐L1 promotes bacterial invasion by altering the dynamics of the actin cytoskeleton

Invasion of eukaryotic target cells by pathogenic bacteria requires extensive remodelling of the membrane and actin cytoskeleton. Here we show that the remodelling process is regulated by the ubiquitin C‐terminal hydrolase UCH‐L1 that promotes the invasion of epithelial cells by Listeria monocytogenes and Salmonella enterica. Knockdown of UCH‐L1 reduced the uptake of both bacteria, while expression of the catalytically active enzyme promoted efficient internalization in the UCH‐L1‐negative HeLa cell line. The entry of L. monocytogenes involves binding to the receptor tyrosine kinase Met, which leads to receptor phosphorylation and ubiquitination. UCH‐L1 controls the early membrane‐associated events of this triggering cascade since knockdown was associated with altered phosphorylation of the c‐cbl docking site on Tyr1003, reduced ubiquitination of the receptor and altered activation of downstream ERK1/2‐ and AKT‐dependent signalling in response to the natural ligand Hepatocyte Growth Factor (HGF). The regulation of cytoskeleton dynamics was further confirmed by the induction of actin stress fibres in HeLa expressing the active enzyme but not the catalytic mutant UCH‐L1_C90S. These findings highlight a previously unrecognized involvement of the ubiquitin cycle in bacterial entry. UCH‐L1 is highly expressed in malignant cells that may therefore be particularly susceptible to invasion by bacteria‐based drug delivery systems.