Bi-allelic variants in WNT7B disrupt the development of multiple organs in humans.

BACKGROUND: Pulmonary hypoplasia, Diaphragmatic anomalies, Anophthalmia/microphthalmia and Cardiac defects delineate the PDAC syndrome. We aim to identify the cause of PDAC syndrome in patients who do not carry pathogenic variants in RARB and STRA6, which have been previously associated with this disorder. METHODS: We sequenced the exome of patients with unexplained PDAC syndrome and performed functional validation of candidate variants. RESULTS: We identified bi-allelic variants in WNT7B in fetuses with PDAC syndrome from two unrelated families. In one family, the fetus was homozygous for the c.292C>T (p.(Arg98*)) variant whereas the fetuses from the other family were compound heterozygous for the variants c.225C>G (p.(Tyr75*)) and c.562G>A (p.(Gly188Ser)). Finally, a molecular autopsy by proxy in a consanguineous couple that lost two babies due to lung hypoplasia revealed that both parents carry the p.(Arg98*) variant. Using a WNT signalling canonical luciferase assay, we demonstrated that the identified variants are deleterious. In addition, we found that wnt7bb mutant zebrafish display a defect of the swimbladder, an air-filled organ that is a structural homolog of the mammalian lung, suggesting that the function of WNT7B has been conserved during evolution for the development of these structures. CONCLUSION: Our findings indicate that defective WNT7B function underlies a form of lung hypoplasia that is associated with the PDAC syndrome, and provide evidence for involvement of the WNT-ß-catenin pathway in human lung, tracheal, ocular, cardiac, and renal development.

Mutations in conserved residues of the C. elegans microRNA Argonaute ALG-1 identify separable functions in ALG-1 miRISC loading and target repression.

microRNAs function in diverse developmental and physiological processes by regulating target gene expression at the post-transcriptional level. ALG-1 is one of two Caenorhabditis elegans Argonautes (ALG-1 and ALG-2) that together are essential for microRNA biogenesis and function. Here, we report the identification of novel antimorphic (anti) alleles of ALG-1 as suppressors of lin-28(lf) precocious developmental phenotypes. The alg-1(anti) mutations broadly impair the function of many microRNAs and cause dosage-dependent phenotypes that are more severe than the complete loss of ALG-1. ALG-1(anti) mutant proteins are competent for promoting Dicer cleavage of microRNA precursors and for associating with and stabilizing microRNAs. However, our results suggest that ALG-1(anti) proteins may sequester microRNAs in immature and functionally deficient microRNA Induced Silencing Complexes (miRISCs), and hence compete with ALG-2 for access to functional microRNAs. Immunoprecipitation experiments show that ALG-1(anti) proteins display an increased association with Dicer and a decreased association with AIN-1/GW182. These findings suggest that alg-1(anti) mutations impair the ability of ALG-1 miRISC to execute a transition from Dicer-associated microRNA processing to AIN-1/GW182 associated effector function, and indicate an active role for ALG/Argonaute in mediating this transition.

The slicing activity of miRNA-specific Argonautes is essential for the miRNA pathway in C. elegans.

Among the set of Argonautes proteins encoded by metazoan genomes, some have conserved amino acids important for catalytic or slicing activity. The functional significance of these residues in microRNA (miRNA)-specific Argonautes in animals is still unclear since miRNAs do not induce site-specific cleavage of targeted messenger RNAs (mRNAs), unlike small interfering RNAs (siRNAs). Here, we report that miRNA-specific ALG-1 and ALG-2 Argonautes from Caenorhabditis elegans possess the slicing activity normally implicated in the siRNA-silencing pathway. We also find that ALG-1/2 can bind and use a Dicer-processed miRNA duplex to target mRNAs, suggesting an ability to displace RNA strands. Importantly, the slicing activity of ALG-1 or ALG-2 is essential for the miRNA pathway in vivo, as shown by the accumulation of truncated miRNA precursors and altered miRNA-induced silencing complex (miRISC) formation. Taken together, our data demonstrate that the slicing activity of Argonautes contributes to a new and unexpected step in the canonical miRNA pathway that occurs prior to miRISC loading in animals.

The Probiotic Strain Lactobacillus acidophilus CL1285 Reduces Fat Deposition and Oxidative Stress and Increases Lifespan in Caenorhabditis elegans.

Caenorhabditis elegans was recently shown to be a powerful model for studying and identifying probiotics with specific functions. Lactobacillus acidophilus CL1285, Lacticaseibacillus casei LBC80R, and Lacticaseibacillus rhamnosus CLR2, which are three bacteria that were marketed by Bio-K+, were evaluated using the nematode C. elegans to study fat accumulation, lifespan, and resistance to oxidative stress. Although the general effects of probiotics in terms of protection against oxidative stress were highlighted, the CL1285 strain had an interesting and specific feature, namely its ability to prevent fat accumulation in nematodes; this effect was verified by both the Oil Red and Nile Red methods. This observed phenotype requires daf-16 and is affected by glucose levels. In addition, in a daf-16- and glucose-dependent manner, CL1285 extended the lifespan of C. elegans; this effect was unique to CL1285 and not found in the other L. acidophilus subtypes in this study. Our findings indicate that L. acidophilus CL1285 impacts fat/glucose metabolism in C. elegans and provides a basis to further study this probiotic, which could have potential health benefits in humans and/or in mammals.

The ribosomal protein RACK1 is required for microRNA function in both C. elegans and humans.

Despite the importance of microRNAs (miRNAs) in gene regulation, it is unclear how the miRNA-Argonaute complex--or miRNA-induced silencing complex (miRISC)--can regulate the translation of their targets in such diverse ways. We demonstrate here a direct interaction between the miRISC and the ribosome by showing that a constituent of the eukaryotic 40S subunit, receptor for activated C-kinase (RACK1), is important for miRNA-mediated gene regulation in animals. In vivo studies demonstrate that RACK1 interacts with components of the miRISC in nematodes and mammals. In both systems, the alteration of RACK1 expression alters miRNA function and impairs the association of the miRNA complex with the translating ribosomes. Our data indicate that RACK1 can contribute to the recruitment of miRISC to the site of translation, and support a post-initiation mode of miRNA-mediated gene repression.

Screening and Characterization of Some Lactobacillaceae for Detection of Cholesterol-Lowering Activities.

Dyslipidemia, specifically abnormal levels of low-density lipoprotein cholesterol (LDL-C), is an important risk factor of cardiovascular disease. Evidence showing the promising abilities of probiotics to lower total cholesterol or LDL-C has, however, not yet convinced experts to recommend probiotic bacteria as treatment for blood lipid management. Therefore, there are opportunities for the development of new efficient cholesterol-lowering probiotics. Bile salt hydrolase (BSH) and feruloyl esterase (FAE) are bacterial enzymes proposed to explain the cholesterol-lowering capacity of some bacteria and have both been shown to be responsible for lipid reduction in vivo. Here, in order to select for cholesterol-lowering bacteria, 70 strains related to Lactobacillaceae were screened for BSH and FAE activities. Based on this two-way screening approach, two bacteria were selected and assessed for their capacity to assimilate cholesterol in vitro, another suggested mechanism. Lactobacillus acidophilus CL1285 showed BSH and FAE activity as well as capacity to assimilate cholesterol in vitro. Lactiplantibacillus plantarum CHOL-200 exhibited BSH activity and ability to assimilate cholesterol. These properties observed in vitro make both strains good probiotic candidates for the management of dyslipidemia. Further investigation is needed to assess their ability to reduce blood cholesterol in human trial.

Quantitative trait loci (QTL) study identifies novel genomic regions associated to Chiari-like malformation in Griffon Bruxellois dogs.

Chiari-like malformation (CM) is a developmental abnormality of the craniocervical junction that is common in the Griffon Bruxellois (GB) breed with an estimated prevalence of 65%. This disease is characterized by overcrowding of the neural parenchyma at the craniocervical junction and disturbance of cerebrospinal fluid (CSF) flow. The most common clinical sign is pain either as a direct consequence of CM or neuropathic pain as a consequence of secondary syringomyelia. The etiology of CM remains unknown but genetic factors play an important role. To investigate the genetic complexity of the disease, a quantitative trait locus (QTL) approach was adopted. A total of 14 quantitative skull and atlas measurements were taken and were tested for association to CM. Six traits were found to be associated to CM and were subjected to a whole-genome association study using the Illumina canine high density bead chip in 74 GB dogs (50 affected and 24 controls). Linear and mixed regression analyses identified associated single nucleotide polymorphisms (SNPs) on 5 Canis Familiaris Autosomes (CFAs): CFA2, CFA9, CFA12, CFA14 and CFA24. A reconstructed haplotype of 0.53 Mb on CFA2 strongly associated to the height of the cranial fossa (diameter F) and an haplotype of 2.5 Mb on CFA14 associated to both the height of the rostral part of the caudal cranial fossa (AE) and the height of the brain (FG) were significantly associated to CM after 10 000 permutations strengthening their candidacy for this disease (P = 0.0421, P = 0.0094 respectively). The CFA2 QTL harbours the Sall-1 gene which is an excellent candidate since its orthologue in humans is mutated in Townes-Brocks syndrome which has previously been associated to Chiari malformation I. Our study demonstrates the implication of multiple traits in the etiology of CM and has successfully identified two new QTL associated to CM and a potential candidate gene.

Function, targets, and evolution of Caenorhabditis elegans piRNAs.

Piwi-interacting RNAs (piRNAs) are small RNAs required to maintain germline integrity and fertility, but their mechanism of action is poorly understood. Here we demonstrate that Caenorhabditis elegans piRNAs silence transcripts in trans through imperfectly complementary sites. Target silencing is independent of Piwi endonuclease activity or "slicing." Instead, piRNAs initiate a localized secondary endogenous small interfering RNA (endo-siRNA) response. Endogenous protein-coding gene and transposon transcripts exhibit Piwi-dependent endo-siRNAs at sites complementary to piRNAs and are derepressed in Piwi mutants. Genomic loci of piRNA biogenesis are depleted of protein-coding genes and tend to overlap the start and end of transposons in sense and antisense, respectively. Our data suggest that nematode piRNA clusters are evolving to generate piRNAs against active mobile elements. Thus, piRNAs provide heritable, sequence-specific triggers for RNA interference in C. elegans.