Functional Characterisation of Bile Metagenome: Study of Metagenomic Dark Matter

Gallbladder metagenome involves a wide range of unidentified sequences comprising the so-called metagenomic dark matter. Therefore, this study aimed to characterise three gallbladder metagenomes and a fosmid library with an emphasis on metagenomic dark matter fraction. For this purpose, a novel data analysis strategy based on the combination of remote homology and molecular modelling has been proposed. According to the results obtained, several protein functional domains were annotated in the metagenomic dark matter fraction including acetyltransferases, outer membrane transporter proteins, membrane assembly factors, DNA repair and recombination proteins and response regulator phosphatases. In addition, one deacetylase involved in mycothiol biosynthesis was found in the metagenomic dark matter fraction of the fosmid library. This enzyme may exert a protective effect in Actinobacteria against bile components exposure, in agreement with the presence of multiple antibiotic and multidrug resistance genes. Potential mechanisms of action of this novel deacetylase were elucidated by molecular simulations, highlighting the role of histidine and aspartic acid residues. Computational pipelines presented in this work may be of special interest to discover novel microbial enzymes which had not been previously characterised.

Fragile X family members have important and non-overlapping functions

The fragile X family of genes encodes a small family of RNA binding proteins including FMRP, FXR1P and FXR2P that were identified in the 1990s. All three members are encoded by 17 exons and show alternative splicing at the 3′ ends of their respective transcripts. They share significant homology in the protein functional domains, including the Tudor domains, the nuclear localization sequence, a protein-protein interaction domain, the KH1 and KH2 domains and the nuclear export sequence. Fragile X family members are found throughout the animal kingdom, although all three members are not consistently present in species outside of mammals: only two family members are present in the avian species examined, Gallus gallus and Taeniopygia guttata, and in the frog Xenopus tropicalis. Although present in many tissues, the functions of the fragile X family members differ, which are particularly evident in knockout studies performed in animals. The fragile X family members play roles in normal neuronal function and in the case of FXR1, in muscle function.

TRANSCRIPT ANNOTATION PRIORITIZATION AND SCREENING SYSTEM (TrAPSS) FOR MUTATION SCREENING

When searching for disease-causing mutations with polymerase chain reaction (PCR)-based methods, candidate genes are usually screened in their entirety, exon by exon. Genomic resources (i.e. www.ncbi.nih.gov, www.ensembl.org, and genome.ucsc.edu) largely support this paradigm for mutation screening by making it easy to view and access sequence data associated with genes in their genomic context. However, the administrative burden of conducting mutation screening in potentially hundreds of genes and thousands of exons in thousands of patients is significant, even with the use of public genome resources. For example, the manual design of oligonucleotide primers for all exons of the 10 Leber's congenital amaurosis (LCA) genes (149 exons) represents a significant information management challenge. The Transcript Annotation Prioritization and Screening System (TrAPSS) is designed to accelerate mutation screening by (1) providing a gene-based local cache of candidate disease genes in a genomic context, (2) automating tasks associated with optimizing candidate disease gene screening and information management, and (3) providing the implementation of an algorithmic technique to utilize large amounts of heterogeneous genome annotation (e.g. conserved protein functional domains) so as to prioritize candidate genes.

A detailed mutagenesis study of flavivirus cross-reactive epitopes using West Nile virus-like particles

Human flavivirus infections elicit virus species-specific and cross-reactive immune responses. The flavivirus envelope (E) glycoprotein is the primary antigen inducing protective immunity; however, the presence of cross-reactive antibodies in human sera creates problems for serodiagnosis. Using a West Nile virus-like particle system, we performed mutagenesis across all three E protein functional domains to identify epitope determinants for a panel of monoclonal antibodies (mAbs) raised against different flaviviruses and exhibiting diverse patterns of cross-reactivity. Residues within the highly conserved fusion peptide were the only epitope determinants identified and were important not only for broadly cross-reactive mAbs recognizing all of the medically important flavivirus serocomplexes, but also for less-broad, complex-reactive mAbs. Moreover, different substitutions at specific fusion peptide residues produced highly variable effects on antibody reactivity and virus-like particle secretion. These results support and extend the conclusion that the fusion peptide region constitutes an immunodominant epitope stimulating antibodies with diverse patterns of cross-reactivity.

MUNC13–4 Mutations in Patients with Hemophagocytic Lymphohistiocytosis Are Scattered over the Functional Domains of the Protein.

Mutations of PRF1 and MUNC13–4 genes, involved with cellular cytotoxicity, are associated with the familial form of Hemophagocytic Lymphohistiocytosis (HLH) type 2 (FHL2) and 3 (FHL3). In all patients with HLH, in which PRF1 mutations had been excluded, we screened MUNC13–4 mutations. The 32 exons and their proximal flanking regions of MUNC13–4 gene were sequenced by “cycle sequencing” approach (BigDye Terminator, Applied Biosystems). Of 60 patients, 21 had MUNC13–4 mutations. 5 were reported mutations: 753+1G>T, 817C>T (R273X); 1389+1G>A; 1822del 12 bp (del V608-A611); 2346–9delGGAG (R782FsX12). Of 16 novel mutations 2 (2212C>T and 2650C>T) introduce a stop codon (at Q738 and Q884); 4 cause frameshift: 441delA (P147fsX14), 532delC (Q178fsX70), 3082delC (L1028fsX), and 3226insG (H1076fsX51). Of 8 missense mutations [175G>A (A59T), 419T>C (I140T), 610A>G (M204B), 1241G>T (R414L), 1847A>G (E616G), 2039G>C (R680P), 2570T>G (F857C), 2782C>T (R928C)], 6 fall within the protein functional domains; each of the 2 falling outside was associated with 2 additional pathogenic mutations. The remaining novel 1992+5 G>A and 2448insC −12 fall outside the coding region but are close enough to induce alternative splicing. We identified polymorphisms 279C>T and 3198A>G. The 2599A>G (K867E) transition was found in several unrelated families, including one homozygous parent. To assess its frequency we studied 50 consecutive newborns, of which 64% were heterozygous. To understand the effects of the mutations we analysed Munc13–4 protein expression from CTL and/or NK cells by western blot using an antibody raised against amino acids 1–262. Trace amounts of protein could be detected only in 5 patients. Analysis of granule polarisation in 2 patients with trace amounts of Munc13–4 protein showed many more docked granules visible than in controls, consistent with a block in granule secretion in these patients. Median age at diagnosis of FHL3 was 6.5 months, but 8/21 (38%) patients were diagnosed when older than 5 years, with one young adult of 18 years. CNS disease was present in 10 patients; NK was markedly reduced or absent in all patients tested. MUNC13–4 mutations were found in 35% of our HLH patients non type-2. Mutations were almost entirely different from those reported so far and scattered over different exons, but in far most cases they fall within the protein functional domain. Since these patients may develop the disease during adolescence or even later on, not only pediatric but also as adults hematologists should include FHL-2 and 3 in the differential diagnosis of children and young adults with fever, cytopenia, splenomegaly and hypercytokinemia.