Structural organization of the 5' region of the thyroglobulin gene. Evidence for intron loss and "exonization" during evolution

More than one third of thyroglobulin (1190 residues out of 2750) is made of one peptide motif repeated ten times in tandem. Segments unrelated to the motif interrupt this structure at various places. The corresponding gene region, which extends over 40 x 10(3) bases, was studied in detail. All exon borders and exon/intron junctions were localized precisely and sequenced, and their positions were correlated with the repetitive organization of the protein. When intron positions were compiled on a consensus sequence of all repeats, three categories of introns were observed. Except between repeats numbers 5 and 6, an intron was invariably found within the Cys codon making the limit of each motif. This category of intron most probably reflects the serial duplication events responsible for the evolution of this region of the gene. All other introns, except no. 2, are found at positions were the repetitive structure is disrupted by "inserted" peptides. We present the hypothesis that this second category of introns was already present in the original unit before the first duplication. Thereafter, they would have experienced either complete loss (some units do not contain any intron) or partial or total exonization, resulting in the slipping of intronic material into coding sequence. Intron no. 2, finally, separates motif no. 1 at a position on the boundary between two segments presenting sequence homology. This last type of intron probably reflects an initial duplication event at the origin of a primordial thyroglobulin gene motif. With all these characteristics, the thyroglobulin gene is presented as a paradigm for the analysis of the fate of introns in gene evolution.     

Use of Caenorhabditis elegans G{alpha}q chimeras to detect G-protein-coupled receptor signals

G-protein-coupled receptors (GPCRs) activate heterotrimeric G-proteins (G(i)-, G(s)-, G(q)-, or G(12)-like) to generate specific intracellular responses, depending on the receptor/G-protein coupling. The aim was to enable a majority of GPCRs to generate a predetermined output by signaling through a single G-protein-supported pathway. The authors focused on calcium responses as the output, then engineered Galpha(q) to promote promiscuous receptor interactions. Starting with a human Galpha(q) containing 5 Galpha(z) residues in the C-terminal receptor recognition domain (hGalpha(q/z5)), they evaluated agonist-stimulated calcium responses for 33 diverse GPCRs (G(i)-, G(s)-, and G(q)-coupled) and found 20 of 33 responders. In parallel, they tested Caenorhabditis elegans Galpha(q) containing 5 or 9 C-terminal Galpha(z) residues (cGalpha(q/z5), cGalpha(q/z9)). Signal detection was enhanced with cGalpha(q/z5) and cGalpha(q/z9) (yielding 25/33 and 26/33 responders, respectively). In a separate study of Galpha(s)-coupled receptors, the authors compared hGalpha(q/s5) versus hGalpha(q/s9), cGalpha(q/s9), andcGalphaq/s21 and observed optimal function with cGalpha(q/s9). Cotransfection of an engineered Galpha(q) "cocktail" (cGalpha(q/z5) plus cGalpha(q/s9)) provided a powerful and efficient screening platform. When the chimeras included N-terminal myristoylation sites (to promote membrane localization), calcium responses were sustained or improved, depending on the receptor. This approach toward a "universal functional assay" is particularly useful for orphan GPCRs whose signaling pathways are unknown.     

The −689/+197 region of the maize protease inhibitor gene directs high level,wound-inducible expression of the cry1B gene which protects transgenic rice plants from stemborer attack

To investigate the activity of the regulatory region of the maize (Zea mays L.) proteinase inhibitor (mpi) gene, we transferred into rice (Oryza sativa L.) plants the –689/+197 (C1) fragment of the mpi genomic clone fused to either theuidA gene or a synthetic Bacillus thuringiensis cry1B gene. Although uidA and cry1B encode very different proteins consistent results were obtained from their respective histochemical and fluorometric and immunoblot detections in T₃ transgenic rice lines. In response to mechanical wounding, a 4–5 fold increase in GUS activity and a Cry1B accumulation reaching 0.1–0.2% of total soluble proteins were observed from basal and undetectable levels respectively in leaf tissue. The establishment of the time-course of wound response in both systems revealed a maximum induction level 12–16 h after treatment. From both systems we also deduced that the C1 region is not active in pollen and seed endosperm. Three independent transformation events expressing cry1B under the control of the C1 region exhibited protection against striped stem borer damage and showed 100% mortality of second instar larvae 8 days after release. These results illustrate the first evidence that wound-inducible expression of a Bacillus thuringiensis endotoxin gene affords full protection to transgenic rice plants.     

Electrocardiographic Screening for Prolonged QT Interval to Reduce Sudden Cardiac Death in Psychiatric Patients: A Cost-Effectiveness Analysis

ImportanceSudden cardiac death is a leading cause of mortality in psychiatric patients. Long QT (LQT) is common in this population and predisposes to Torsades-de-Pointes (TdP) and subsequent mortality.ObjectiveTo estimate the cost-effectiveness of electrocardiographic screening to detect LQT in psychiatric inpatients.ResultsIn the base-case scenario, the numbers of patients needed to screen were 1128 and 2817 to avoid one TdP and one death, respectively. The ICER of systematic ECG screening was $8644 (95%CI, 3144-82 498) per QALY. The probability of cost-effectiveness was 96% at a willingness-to-pay of $50 000 for one QALY. In sensitivity analyses, results were sensitive to the case-fatality of TdP episodes and to the TdP reduction following the diagnosis of LQT.

Conclusion and Relevance

In psychiatric hospitals, performing systematic ECG screening at admission help reduce the number of sudden cardiac deaths in a cost-effective fashion.     

Dual function of CALCOCO2/NDP52 during xenophagy

During xenophagy, pathogens are selectively targeted by autophagy receptors to the autophagy machinery for their subsequent degradation. In infected cells, the autophagy receptor CALCOCO2/NDP52 targets Salmonella Typhimurium to the phagophore membrane by concomitantly interacting with LC3C and binding to ubiquitinated cytosolic bacteria or to LGALS8/GALECTIN 8 adsorbed on damaged vacuoles that contain bacteria. We recently reported that in addition, CALCOCO2 is also necessary for the maturation step of Salmonella Typhimurium-containing autophagosomes. Interestingly, the role of CALCOCO2 in maturation is independent of its role in targeting, as these functions rely on distinct binding domains and protein partners. Indeed, to mediate autophagosome maturation CALCOCO2 binds on the one hand to LC3A, LC3B, or GABARAPL2, and on the other hand to MYO6/MYOSIN VI, whereas the interaction with LC3C is dispensable. Therefore, the autophagy receptor CALCOCO2 plays a dual function during xenophagy first by targeting bacteria to nascent autophagosomes and then by promoting autophagosome maturation in order to destroy bacteria.Xenophagy is the process referring to the selective degradation of intracellular microorganisms by autophagy. Xenophagy is a very potent intrinsic cellular line of defense to fight pathogens and requires first the detection and targeting of microorganisms to growing phagophores prior to autophagosome maturation leading to microbial destruction. The targeting step can be achieved by cytosolic autophagy receptors, which bind on the one hand to the pathogen and on the other hand to LC3, a phagophore membrane-anchored protein. Once entrapped within an autophagosome, bacteria can survive or escape, unless they are rapidly destroyed. Therefore, autophagosome maturation allows the discharge of lysosomal enzymes in autolysosomes, allowing destruction of the bacteria. It is, however, not well known how autophagosomes mature, especially in the context of xenophagy. Recently, the endosomal membrane-bound protein TOM1 and the dynein motor MYO6 have been both shown to be implicated in the transport of endosomes into the vicinity of autophagosomes in order to ensure fusion of autophagosomes with vesicles of the endo/lysosomal pathway. Moreover, the concomitant absence of 3 autophagy receptors, CALCOCO2, TAX1BP1/T6BP, and OPTN/OPTINEURIN, impairs autophagosome biogenesis and maturation. As CALCOCO2 was already shown to have a MYO6 binding domain, we wondered whether CALCOCO2 could also be implicated in autophagosome maturation per se to promote bacterial degradation.We first observed that the binding site of CALCOCO2 to MYO6 was required for cells to control Salmonella Typhimurium intracellular growth. Nevertheless, when the binding of CALCOCO2 to MYO6 was abolished, bacteria were still efficiently targeted to autophagosomes, but yet still able to replicate to levels similar to the one observed in CALCOCO2-depleted cells. Strikingly, in noninfected cells the absence of CALCOCO2 perturbs the autophagy flux, resulting in a strong accumulation of autophagosomes, suggesting a positive role for CALCOCO2 in the autophagosome-lysosome fusion process. Surprisingly, we found that CALCOCO2 binding to LC3C, through its noncanonical LC3 interacting region (CLIR), is not involved in the maturation of autophagosomes. Instead, we identified another motif in the primary sequence of CALCOCO2, which mediates binding to at least LC3A, LC3B, and GABARAPL2 (but not LC3C). We referred to this motif as “LIR-like” as it differs from the canonical LIR motif by the absence of a hydrophobic residue in position X₃. This LIR-like motif was necessary for autophagosome maturation, along with the domain of CALCOCO2 responsible for its binding to MYO6. Eventually, mutation of this LIR-like motif also resulted in an increased Salmonella Typhimurium intracellular proliferation, whereas bacteria were still efficiently targeted within nondegradative autophagosomes. Interestingly, the absence of the autophagy receptor OPTN also led to the accumulation of nondegradative autophagosomes, suggesting that other autophagy receptors could share CALCOCO2 dual functions in xenophagy.Having autophagy receptors ensuring both targeting and degradation of pathogens could be an important evolutionary advantage against infections. Indeed, this mechanism could help to reduce the delay necessary for maturation, thus avoiding adaptation of the pathogen to its new environment (as proposed for Coxiella burnetti, Listeria monocytogenes, and Legionella pneumophila) or its escape from the autophagosome. Conversely, pathogens could avoid autophagy entrapment or autophagic degradation by targeting CALCOCO2 or any other autophagy receptors, which could play similar roles. For instance Chikungunya virus was reported to target CALCOCO2 in human cells leading to increased virus replication. Nevertheless, redundancy among autophagy receptors could also ensure a selective immune advantage against pathogens targeting any one of these receptors.Our results and those from others suggest for now that CALCOCO2 serves as a docking platform for MYO6-bound endosomes, thus facilitating autophagosome maturation (Fig. 1). How this action is coordinated with CALCOCO2 directing pathogens to the phagophore membranes remains unclear. During xenophagy against Salmonella Typhimurium, CALCOCO2 interaction first with LC3C is necessary to further recruit other ATG8 orthologs and ensure the final degradation of bacteria. Since the LIR-like motifs bind several ATG8s, whereas the CLIR motif only mediates binding to LC3C, it is possible that binding of CALCOCO2 to LC3C induces conformational changes and uncovers the LIR-like motif that can be then engaged with other ATG8 orthologs to trigger autophagosome maturation. Moreover, it is still unclear whether the action of CALCOCO2 in autophagosome maturation is coordinated with other partners, such as STX17/SYNTAXIN 17, which is recruited on the external membrane of autophagosomes and regulate fusion with lysosomes. Open in a separate windowFigure 1.Schematic model for the dual role of CALCOCO2 in xenophagy. CALCOCO2 targets bacteria to the phagophore through its LC3C binding site (CLIR motif), and, independently, regulates autophagosome maturation through its LC3A, LC3B, or GABARAPL2 binding site (LIR-like motif) and its MYO6 interacting region.Our findings reveal a new role for the autophagy receptor CALCOCO2 in autophagosome maturation, unravelling another function for CALCOCO2 in cell autonomous defense against pathogens: CALCOCO2 not only targets pathogens to phagophore membranes, but also regulates subsequent maturation of pathogen-containing autophagosomes, thus assuring efficient degradation of autophagy-targeted pathogens.     

Is local selection so widespread in river organisms? Fractal geometry of river networks leads to high bias in outlier detection

Identifying local adaptation is crucial in conservation biology to define ecotypes and establish management guidelines. Local adaptation is often inferred from the detection of loci showing a high differentiation between populations, the so‐called F_ST outliers. Methods of detection of loci under selection are reputed to be robust in most spatial population models. However, using simulations we showed that F_ST outlier tests provided a high rate of false‐positives (up to 60%) in fractal environments such as river networks. Surprisingly, the number of sampled demes was correlated with parameters of population genetic structure, such as the variance of F_STs, and hence strongly influenced the rate of outliers. This unappreciated property of river networks therefore needs to be accounted for in genetic studies on adaptation and conservation of river organisms.     

The impact of homologous recombination on the generation of diversity in bacteria

The imprint of demographic and selective processes on bacterial population structure needs to be evaluated as deviation from the expectations of an appropriate null neutral model. We explore the impact of varying the population mutation and recombination rates theta and rho on ideal populations, using a recently developed model of neutral drift at multiple loci. This model may be fitted to experimental data to provide estimates of these parameters, and we do so for seven bacterial species (Neisseria meningitidis, Streptococcus pneumoniae, Streptococcus pyogenes, Staphylococcus aureus, Helicobacter pylori, Burkholderia pseudomallei and Bacillus cereus), illustrating that bacterial species vary extensively in these fundamental parameters. Historically, the influence of recombination has often been estimated through its influence on the Index of Association I(A). We show that this may be relatively insensitive to changes in either mutation or recombination rates. It is known that biased sampling can lead to artificially high estimates of I(A). We therefore provide a method of precisely separating the effects of such bias and true linkage between alleles. We also demonstrate that by fitting the neutral model to experimental data, more informative and precise estimates of the relative roles of recombination and mutation may be obtained.