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Phosphoribosylaminoimidazole carboxylase, phosphoribosylaminoimidazole succinocarboxamide synthetase (PAICS) encodes an enzyme that catalyzes de novo purine biosynthesis. Although PAICS has been implicated as a potential therapeutic target in several cancers, its clinical and prognostic significance in colorectal cancer (CRC) is not fully understood. To elucidate the roles of PAICS in CRC, we investigated PAICS expression in four cohorts consisting of a total of 1659 samples based on quantitative RT-PCR, microarray and RNA-seq analysis. Despite upregulated PAICS levels in tumor compared to those of normal mucosa, we found a decreasing trend of PAICS expression during tumor progression and metastasis. We conducted immunohistochemistry on 252 specimens, showing that PAICS protein was strongly expressed in the majority of CRCs, but not in adjacent mucosa. Notably, 29.0% of tumors lacked PAICS staining, and PAICS-negative expression in tumor had significant prognostic impact on poor cancer-specific survival in stage III CRC. Correspondingly, decreased levels of PAICS transcript were also correlated with poor relapse-free survival particularly in stage III patients, and this finding was robustly confirmed in three microarray datasets of a total of 802 stage II-III patients. Bioinformatics analysis of CRC tissues and cell lines consistently indicated a correlation between decreased PAICS expression and copy number loss of chromosome arm 4q. In conclusion, our results suggest that PAICS expression is downregulated during tumor progression due to genetic deletion of chromosome 4q in microsatellite stable but chromosomally unstable tumors. Furthermore, decreased expression of PAICS transcript or loss of PAICS protein may provide prognostic stratification for postoperative patients with stage III CRC.

Avian influenza virus H9N2 has been endemic in birds in the Middle East, in particular in Egypt with multiple cases of human infections since 1998. Despite concerns about the pandemic threat posed by H9N2, little is known about the biological properties of H9N2 in this epicentre of infection. Here, we investigated the evolutionary dynamics of H9N2 in the Middle East and identified phylogeny-associated PB2 mutations that acted cooperatively to increase H9N2 replication/transcription in human cells. The accumulation of PB2 mutations also correlated with an increase in H9N2 virus growth in the upper and lower airways of mice and in virulence. These mutations clustered on a solvent-exposed region in the PB2-627 domain in proximity to potential interfaces with host factors. These PB2 mutations have been found at high prevalence during evolution of H9N2 in the field, indicating that they have provided a selective advantage for viral adaptation to infect poultry. Therefore, continuous prevalence of H9N2 virus in the Middle East has generated a far more fit or optimized replication phenotype, leading to an expanded viral host range, including to mammals, which may pose public health risks beyond the current outbreaks.

A major determinant in the change of the avian influenza virus host range to humans is the E627K substitution in the PB2 polymerase protein. However, the polymerase activity of avian influenza viruses with a single PB2-E627K mutation is still lower than that of seasonal human influenza viruses, implying that avian viruses require polymerase mutations in addition to PB2-627K for human adaptation. Here, we used a database search of H5N1 clade 2.2.1 virus sequences with the PB2-627K mutation to identify other polymerase adaptation mutations that have been selected in infected patients. Several of the mutations identified acted cooperatively with PB2-627K to increase viral growth in human airway epithelial cells and mouse lungs. These mutations were in multiple domains of the polymerase complex other than the PB2-627 domain, highlighting a complicated avian-to-human adaptation pathway of avian influenza viruses. Thus, H5N1 viruses could rapidly acquire multiple polymerase mutations that function cooperatively with PB2-627K in infected patients for optimal human adaptation.

Psychological stress can exacerbate inflammatory bowel disease. However, the mechanisms underlying how psychological stress affects gut inflammation remain unclear. Here, we focused on the relationship between changes in the microbial community of mucosa-associated commensal bacteria (MACB) and mucosal immune responses induced by chronic psychological stress in a murine model of ulcerative colitis. Furthermore, we examined the effect of probiotic treatment on exacerbated colitis and MACB composition changes induced by chronic psychological stress. Repeated water avoidance stress (rWAS) in B6-Tcra-/- mice severely exacerbated colitis, which was evaluated by both colorectal tissue weight and histological score of colitis. rWAS treatment increased mRNA expression of UCN2 and IFN-γ in large intestinal lamina propria mononuclear cells (LI-LPMC). Interestingly, exacerbated colitis was associated with changes in the microbial community of MACB, specifically loss of bacterial species diversity and an increase in the component ratio of Clostridium, revealed by 16S rRNA gene amplicon analysis. Finally, the oral administration of a probiotic Lactobacillus strain was protective against the exacerbation of colitis and was associated with a change in the bacterial community of MACB in rWAS-exposed Tcra-/- mice. Taken together, these results suggested that loss of species diversity in MACB might play a key role in exacerbated colitis induced by chronic psychological stress. In addition, probiotic treatment may be used as a tool to preserve the diversity of bacterial species in MACB and alleviate gut inflammation induced by psychological stress.

Phagocytes such as dendritic cells and macrophages, which are distributed in the small intestinal mucosa, play a crucial role in maintaining mucosal homeostasis by sampling the luminal gut microbiota. However, there is limited information regarding microbial uptake in a steady state. We investigated the composition of murine gut microbiota that is engulfed by phagocytes of specific subsets in the small intestinal lamina propria (SILP) and Peyer's patches (PP). Analysis of bacterial 16S rRNA gene amplicon sequences revealed that: 1) all the phagocyte subsets in the SILP primarily engulfed Lactobacillus (the most abundant microbe in the small intestine), whereas CD11bhi and CD11bhiCD11chi cell subsets in PP mostly engulfed segmented filamentous bacteria (indigenous bacteria in rodents that are reported to adhere to intestinal epithelial cells); and 2) among the Lactobacillus species engulfed by the SILP cell subsets, L. murinus was engulfed more frequently than L. taiwanensis, although both these Lactobacillus species were abundant in the small intestine under physiological conditions. These results suggest that small intestinal microbiota is selectively engulfed by phagocytes that localize in the adjacent intestinal mucosa in a steady state. These observations may provide insight into the crucial role of phagocytes in immune surveillance of the small intestinal mucosa.

The effect of psychological stress on the gastrointestinal microbiota is widely recognized. Chronic psychological stress may be associated with increased disease activity in inflammatory bowel disease, but the relationships among psychological stress, the gastrointestinal microbiota, and the severity of colitis is not yet fully understood. Here, we examined the impact of 12-week repeated water-avoidance stress on the microbiota of two inbred strains of T cell receptor alpha chain gene knockout mouse (background, BALB/c and C57BL/6) by means of next-generation sequencing of bacterial 16S rRNA genes. In both mouse strains, knockout of the T cell receptor alpha chain gene caused a loss of gastrointestinal microbial diversity and stability. Chronic exposure to repeated water-avoidance stress markedly altered the composition of the colonic microbiota of C57BL/6 mice, but not of BALB/c mice. In C57BL/6 mice, the relative abundance of genus Clostridium, some members of which produce the toxin phospholipase C, was increased, which was weakly positively associated with colitis severity, suggesting that expansion of specific populations of indigenous pathogens may be involved in the exacerbation of colitis. However, we also found that colitis was not exacerbated in mice with a relatively diverse microbiota even if their colonic microbiota contained an expanded phospholipase C-producing Clostridium population. Exposure to chronic stress also altered the concentration of free immunoglobulin A in colonic contents, which may be related to both the loss of bacterial diversity in the colonic microbiota and the severity of the colitis exacerbation. Together, these results suggest that long-term exposure to psychological stress induces dysbiosis in the immunodeficient mouse in a strain-specific manner and also that alteration of microbial diversity, which may be related to an altered pattern of immunoglobulin secretion in the gastrointestinal tract, might play a crucial role in the development of chronic stress-induced colitis.

An enormous number of alternative pre-mRNA splicing patterns in multicellular organisms are coordinately defined by a limited number of regulatory proteins and cis elements. Mutually exclusive alternative splicing should be strictly regulated and is a challenging model for elucidating regulation mechanisms. Here we provide models of the regulation of two sets of mutually exclusive exons, 4a-4c and 7a-7b, of the Caenorhabditis elegans uncoordinated (unc)-32 gene, encoding the a subunit of V0 complex of vacuolar-type H(+)-ATPases. We visualize selection patterns of exon 4 and exon 7 in vivo by utilizing a trio and a pair of symmetric fluorescence splicing reporter minigenes, respectively, to demonstrate that they are regulated in tissue-specific manners. Genetic analyses reveal that RBFOX family RNA-binding proteins ASD-1 and FOX-1 and a UGCAUG stretch in intron 7b are involved in the neuron-specific selection of exon 7a. Through further forward genetic screening, we identify UNC-75, a neuron-specific CELF family RNA-binding protein of unknown function, as an essential regulator for the exon 7a selection. Electrophoretic mobility shift assays specify a short fragment in intron 7a as the recognition site for UNC-75 and demonstrate that UNC-75 specifically binds via its three RNA recognition motifs to the element including a UUGUUGUGUUGU stretch. The UUGUUGUGUUGU stretch in the reporter minigenes is actually required for the selection of exon 7a in the nervous system. We compare the amounts of partially spliced RNAs in the wild-type and unc-75 mutant backgrounds and raise a model for the mutually exclusive selection of unc-32 exon 7 by the RBFOX family and UNC-75. The neuron-specific selection of unc-32 exon 4b is also regulated by UNC-75 and the unc-75 mutation suppresses the Unc phenotype of the exon-4b-specific allele of unc-32 mutants. Taken together, UNC-75 is the neuron-specific splicing factor and regulates both sets of the mutually exclusive exons of the unc-32 gene.

Mixed-lineage-leukemia (MLL) fusion oncogenes are intimately involved in acute leukemia and secondary therapy-related acute leukemia. To understand MLL-rearranged leukemia, several murine models for this disease have been established. However, the mouse leukemia derived from mouse hematopoietic stem cells (HSCs) may not be fully comparable with human leukemia. Here we developed a humanized mouse model for human leukemia by transplanting human cord blood-derived HSCs transduced with an MLL-AF10 oncogene into a supra-immunodeficient mouse strain, NOD/Shi-scid, IL-2Rγ(-/-) (NOG) mice. Injection of the MLL-AF10-transduced HSCs into the liver of NOG mice enhanced multilineage hematopoiesis, but did not induce leukemia. Because active mutations in ras genes are often found in MLL-related leukemia, we next transduced the gene for a constitutively active form of K-ras along with the MLL-AF10 oncogene. Eight weeks after transplantation, all the recipient mice had developed acute monoblastic leukemia (the M5 phenotype in French-American-British classification). We thus successfully established a human MLL-rearranged leukemia that was derived in vivo from human HSCs. In addition, since the enforced expression of the mutant K-ras alone was insufficient to induce leukemia, the present model may also be a useful experimental platform for the multi-step leukemogenesis model of human leukemia.

Pre-mRNAs are often processed in complex patterns in tissue-specific manners to produce a variety of protein isoforms from single genes. However, mechanisms orchestrating the processing of the entire transcript are not well understood. Muscle-specific alternative pre-mRNA processing of the unc-60 gene in Caenorhabditis elegans, encoding two tissue-specific isoforms of ADF/cofilin with distinct biochemical properties in regulating actin organization, provides an excellent in vivo model of complex and tissue-specific pre-mRNA processing; it consists of a single first exon and two separate series of downstream exons. Here we visualize the complex muscle-specific processing pattern of the unc-60 pre-mRNA with asymmetric fluorescence reporter minigenes. By disrupting juxtaposed CUAAC repeats and UGUGUG stretch in intron 1A, we demonstrate that these elements are required for retaining intron 1A, as well as for switching the processing patterns of the entire pre-mRNA from non-muscle-type to muscle-type. Mutations in genes encoding muscle-specific RNA-binding proteins ASD-2 and SUP-12 turned the colour of the unc-60 reporter worms. ASD-2 and SUP-12 proteins specifically and cooperatively bind to CUAAC repeats and UGUGUG stretch in intron 1A, respectively, to form a ternary complex in vitro. Immunohistochemical staining and RT-PCR analyses demonstrate that ASD-2 and SUP-12 are also required for switching the processing patterns of the endogenous unc-60 pre-mRNA from UNC-60A to UNC-60B in muscles. Furthermore, systematic analyses of partially spliced RNAs reveal the actual orders of intron removal for distinct mRNA isoforms. Taken together, our results demonstrate that muscle-specific splicing factors ASD-2 and SUP-12 cooperatively promote muscle-specific processing of the unc-60 gene, and provide insight into the mechanisms of complex pre-mRNA processing; combinatorial regulation of a single splice site by two tissue-specific splicing regulators determines the binary fate of the entire transcript.

Highly pathogenic avian influenza A virus subtype H5N1 is currently widespread in Asia, Europe, and Africa, with 60% mortality in humans. In particular, since 2009 Egypt has unexpectedly had the highest number of human cases of H5N1 virus infection, with more than 50% of the cases worldwide, but the basis for this high incidence has not been elucidated. A change in receptor binding affinity of the viral hemagglutinin (HA) from α2,3- to α2,6-linked sialic acid (SA) is thought to be necessary for H5N1 virus to become pandemic. In this study, we conducted a phylogenetic analysis of H5N1 viruses isolated between 2006 and 2009 in Egypt. The phylogenetic results showed that recent human isolates clustered disproportionally into several new H5 sublineages suggesting that their HAs have changed their receptor specificity. Using reverse genetics, we found that these H5 sublineages have acquired an enhanced binding affinity for α2,6 SA in combination with residual affinity for α2,3 SA, and identified the amino acid mutations that produced this new receptor specificity. Recombinant H5N1 viruses with a single mutation at HA residue 192 or a double mutation at HA residues 129 and 151 had increased attachment to and infectivity in the human lower respiratory tract but not in the larynx. These findings correlated with enhanced virulence of the mutant viruses in mice. Interestingly, these H5 viruses, with increased affinity to α2,6 SA, emerged during viral diversification in bird populations and subsequently spread to humans. Our findings suggested that emergence of new H5 sublineages with α2,6 SA specificity caused a subsequent increase in human H5N1 influenza virus infections in Egypt, and provided data for understanding the virus's pandemic potential.

Borna disease virus (BDV) is a nonsegmented, negative-strand RNA virus that employs several unique strategies for gene expression. The shortest transcript of BDV, X/P mRNA, encodes at least three open reading frames (ORFs): upstream ORF (uORF), X, and P in the 5' to 3' direction. The X is a negative regulator of viral polymerase activity, while the P phosphoprotein is a necessary cofactor of the polymerase complex, suggesting that the translation of X is controlled rigorously, depending on viral replication. However, the translation mechanism used by the X/P polycistronic mRNA has not been determined in detail. Here we demonstrate that the X/P mRNA autogenously regulates the translation of X via interaction with host factors. Transient transfection of cDNA clones corresponding to the X/P mRNA revealed that the X ORF is translated predominantly by uORF-termination-coupled reinitiation, the efficiency of which is upregulated by expression of P. We found that P may enhance ribosomal reinitiation at the X ORF by inhibition of the interaction of the DEAD-box RNA helicase DDX21 with the 5' untranslated region of X/P mRNA, via interference with its phosphorylation. Our results not only demonstrate a unique translational control of viral regulatory protein, but also elucidate a previously unknown mechanism of regulation of polycistronic mRNA translation using RNA helicases.