Meta-analysis Reveals Genome-Wide Significance at 15q13 for Nonsyndromic Clefting of Both the Lip and the Palate,and Functional Analyses Implicate GREM1 As a Plausible Causative Gene

Nonsyndromic orofacial clefts are common birth defects with multifactorial etiology. The most common type is cleft lip, which occurs with or without cleft palate (nsCLP and nsCLO, respectively). Although genetic components play an important role in nsCLP, the genetic factors that predispose to palate involvement are largely unknown. In this study, we carried out a meta-analysis on genetic and clinical data from three large cohorts and identified strong association between a region on chromosome 15q13 and nsCLP (P = 8.13×10⁻¹⁴ for rs1258763; relative risk (RR): 1.46, 95% confidence interval (CI): 1.32–1.61)) but not nsCLO (P = 0.27; RR: 1.09 (0.94–1.27)). The 5 kb region of strongest association maps downstream of Gremlin-1 (GREM1), which encodes a secreted antagonist of the BMP4 pathway. We show during mouse embryogenesis, Grem1 is expressed in the developing lip and soft palate but not in the hard palate. This is consistent with genotype-phenotype correlations between rs1258763 and a specific nsCLP subphenotype, since a more than two-fold increase in risk was observed in patients displaying clefts of both the lip and soft palate but who had an intact hard palate (RR: 3.76, CI: 1.47–9.61, P_diff<0.05). While we did not find lip or palate defects in Grem1-deficient mice, wild type embryonic palatal shelves developed divergent shapes when cultured in the presence of ectopic Grem1 protein (P = 0.0014). The present study identified a non-coding region at 15q13 as the second, genome-wide significant locus specific for nsCLP, after 13q31. Moreover, our data suggest that the closely located GREM1 gene contributes to a rare clinical nsCLP entity. This entity specifically involves abnormalities of the lip and soft palate, which develop at different time-points and in separate anatomical regions.     

Inter‐kingdom signaling — symbiotic yeasts produce semiochemicals that attract their yellowjacket hosts

In recent studies, the yeast species Hanseniaspora uvarum and Lachancea thermotolerans were isolated from the digestive tract of four North American yellowjacket species (Hymenoptera: Vespidae), and attraction of yellowjackets to brewer's yeast, Saccharomyces cerevisiae (all Saccharomycetaceae), growing on fruit powder was demonstrated. We tested the hypothesis that Vespula spp. are attracted to cultures of H. uvarum and L. thermotolerans and their respective volatiles. In field experiments, we found that H. uvarum and L. thermotolerans are attractive to three species of yellowjacket, but only when grown on grape juice‐infused yeast peptone dextrose (YPD) agar. Using gas chromatography‐mass spectrometry, we analyzed the headspace volatiles produced by these yeasts, and field tested an 18‐component yeast synthetic semiochemical blend. This synthetic blend attracted western yellowjackets, Vespula pensylvanica (Saussure), but no other yellowjacket species. Acetic acid or ethanol added to the synthetic blend at biologically relevant doses either had no effect or significantly lowered trap captures. Our results demonstrate that yeast symbionts isolated from the digestive tract of yellowjackets are attractive to their hosts. Further research is needed to identify the volatiles mediating attraction of species other than V. pensylvanica to the yeast cultures.     

Zonal expression of hepatocytic marker enzymes during liver repopulation

Hepatocytes are metabolically specialised cells displaying distinctive gene expression patterns within the liver lobule. Here, we investigate whether pre-cultured adult rat hepatocytes adopt periportal and pericentral enzyme expression following their transplantation into the regenerating rat liver. Isolated primary rat hepatocytes, representing a mixture of both periportal and pericentral origin, lost expression of carbamoyl phosphate synthetase I (CPS I) and cytochrome P450 subtype 2B1 (CYP2B1) in culture as shown by immunofluorescence and Western blot analysis. Accordingly, urea synthesis and CYP2B1 enzyme activity decreased. Hepatocytes from DPPIV (CD26) wild type rats were cultured for 4 and 7 days, and then transplanted into the livers of CD26 deficient rats following prior treatment with retrorsine and partial hepatectomy to drive selective donor cell proliferation. CD26 positive donor cells engrafted in the periportal regions and grew in clusters expanding into the parenchyma as time proceeded. Ten weeks after transplantation, cells derived from donors surrounding the portal veins expressed CPS I, but not CYP2B1. The reverse was true for CD26 positive cells in close proximity to the central veins displaying immunoreactivity to CYP2B1, but no longer to CPS I. Hepatocytes lose their specific marker enzyme expression in culture. After transplantation, donor hepatocytes proliferate in the host parenchyma whilst acquiring the position-specific enzyme expression of the surrounding periportal and pericentral host hepatocytes. These results indicate the high degree of plasticity of gene expression in hepatocytes subjected to a change in microenvironment.     

The ATPase and helicase activities of Prp43p are stimulated by the G‐patch protein Pfa1p during yeast ribosome biogenesis

Prp43p is a RNA helicase required for pre‐mRNA splicing and for the synthesis of large and small ribosomal subunits. The molecular functions and modes of regulation of Prp43p during ribosome biogenesis remain unknown. We demonstrate that the G‐patch protein Pfa1p, a component of pre‐40S pre‐ribosomal particles, directly interacts with Prp43p. We also show that lack of Gno1p, another G‐patch protein associated with Prp43p, specifically reduces Pfa1p accumulation, whereas it increases the levels of the pre‐40S pre‐ribosomal particle component Ltv1p. Moreover, cells lacking Pfa1p and depleted for Ltv1p show strong 20S pre‐rRNA accumulation in the cytoplasm and reduced levels of 18S rRNA. Finally, we demonstrate that Pfa1p stimulates the ATPase and helicase activities of Prp43p. Truncated Pfa1p variants unable to fully stimulate the activity of Prp43p fail to complement the 20S pre‐rRNA processing defect of Δpfa1 cells depleted for Ltv1p. Our results strongly suggest that stimulation of ATPase/helicase activities of Prp43p by Pfa1p is required for efficient 20S pre‐rRNA‐to‐18S rRNA conversion.     

The role of CD14 in neutrophil recruitment within the liver microcirculation during endotoxemia

During Gram-negative sepsis and endotoxemia, CD14 is essential for the recognition of LPS by the TLR4 complex and subsequent generation of systemic inflammation. However, CD14-independent responses to LPS have been reported in vitro and in vivo in selected tissues including the skin. As the liver is a key target organ for neutrophil sequestration and inflammatory pathology during sepsis and endotoxemia, we investigated the role of CD14 in the recruitment of neutrophils into the liver in a mouse model of endotoxemia. Using dynamic in vivo imaging of the liver, we observed that neutrophil recruitment within the sinusoids and post-sinusoidal venules occurred equivalently between LPS-treated wild-type and CD14-knockout mice. Neutrophil recruitment within the liver was completely independent of CD14 regardless of whether it was expressed on cells of hematopoietic or nonhematopoietic origin or in serum as soluble CD14. Whereas CD14 expression was essential for activation of circulating neutrophils and for the development of LPS-induced systemic inflammation (pulmonary neutrophil sequestration, leukopenia, and increased serum proinflammatory cytokine levels), deficiency of CD14 did not limit the adhesion strength of neutrophils in vitro. Furthermore, wild-type and CD14-knockout mice displayed identical deposition of serum-derived hyaluronan-associated protein within liver sinusoids in response to LPS, indicating that the sinusoid-specific CD44/hyaluronan/serum-derived hyaluronan-associated protein-dependent pathway of neutrophil adhesion is activated independently of CD14. Therefore, the liver microcirculation possesses a unique CD14-independent mechanism of LPS detection and activation of neutrophil recruitment.     

Site-specific deletion at the replication origin of the antibiotic resistance factor R1

Summary The recombinant plasmid pRK101 carrying the complete replication origin of the antibiotic resistance factor R1 suffers frequently a deletion of 218 base pairs, removing parts or all of the origin sequence. This deletion seems to occur always when the Pst-E fragment carrying the replication origin is inserted into the cloning vector pBR322 in an orientation where the direction of R1 replication is the same as that of the vector plasmid and frequently when it is inserted in the opposite direction. DNA sequence analysis around the junction site generated by the deletion in three independently isolated deletion mutants reveals that the deletion occurs at a specific site, namely the end of a 22 bp sequence which is repeated almost identically at the other end of a segment of 197 bp. During the deletion one repeat unit is removed whereas the other is retained. The DNA sequence included by the two repeats contains high symmetric structures, i.e. inverted repeats, direct repeats and palindromes which may represent regulatory sites of the origin.