Identification of Dhx15 as a Major Regulator of Liver Development, Regeneration, and Tumor Growth in Zebrafish and Mice.

RNA helicase DHX15 plays a significant role in vasculature development and lung metastasis in vertebrates. In addition, several studies have demonstrated the overexpression of DHX15 in the context of hepatocellular carcinoma. Therefore, we hypothesized that this helicase may play a significant role in liver regeneration, physiology, and pathology. Dhx15 gene deficiency was generated by CRISPR/Cas9 in zebrafish and by TALEN-RNA in mice. AUM Antisense-Oligonucleotides were used to silence Dhx15 in wild-type mice. The hepatocellular carcinoma tumor induction model was generated by subcutaneous injection of Hepa 1-6 cells. Homozygous Dhx15 gene deficiency was lethal in zebrafish and mouse embryos. Dhx15 gene deficiency impaired liver organogenesis in zebrafish embryos and liver regeneration after partial hepatectomy in mice. Also, heterozygous mice presented decreased number and size of liver metastasis after Hepa 1-6 cells injection compared to wild-type mice. Dhx15 gene silencing with AUM Antisense-Oligonucleotides in wild-type mice resulted in 80% reduced expression in the liver and a significant reduction in other major organs. In addition, Dhx15 gene silencing significantly hindered primary tumor growth in the hepatocellular carcinoma experimental model. Regarding the potential use of DHX15 as a diagnostic marker for liver disease, patients with hepatocellular carcinoma showed increased levels of DHX15 in blood samples compared with subjects without hepatic affectation. In conclusion, Dhx15 is a key regulator of liver physiology and organogenesis, is increased in the blood of cirrhotic and hepatocellular carcinoma patients, and plays a key role in controlling hepatocellular carcinoma tumor growth and expansion in experimental models.

Self-delivering mRNA inhibitors of MK2 improve outcomes after spinal cord injury.

Functional recovery and tissue damage after spinal cord injury (SCI) are influenced by secondary damage mechanisms, including inflammation. The inflammatory response after SCI relies on a variety of cell types with both protective and cytotoxic functions. The macrophage derived MAPK-activated protein kinase 2 has been described as a critical regulator of inflammation with detrimental function after SCI. Targeted modification of inflammatory effector molecules after SCI faces challenges of optimal timing, dosage and location of administration. Modified RNA inhibitors, FANA antisense oligonucleotides, are promising inhibitors due to their stability, local penetration of cells and high efficacy in targeted suppression. Here, we describe the use of anti- MAPK-activated protein kinase 2 FANA antisense oligonucleotides in a mouse model of contusional SCI. The most efficient inhibitor was selected with in vitro and in vivo techniques and then applied via intrathecal injections after SCI. This treatment resulted in improved gait applying DigiGait assessments and tissue preservation, indicating the usefulness of the target and inhibition approach.

Differential regulation of the Tor gene homolog drives the red/green pigmentation phenotype in the aphid Myzuspersicae.

In some aphid species, intraspecific variation in body colour is caused by differential carotenoid content: whilst green aphids contain only yellow carotenoids (ß-, γ-, and ß,γ-carotenes), red aphids additionally possess red carotenoids (torulene and 3,4-didehydrolycopene). Unusually, within animals who typically obtain carotenoids from their diet, ancestral horizontal gene transfer of carotenoid biosynthetic genes from fungi (followed by gene duplication), have imbued aphids with the intrinsic gene repertoire necessary to biosynthesise carotenoids. In the pea aphid, Acyrthosiphon pisum a lycopene (phytoene) desaturase gene (Tor) underpins the red/green phenotype, with this locus present in heterozygous form in red individuals but absent in green aphids, resulting in them being unable to convert lycopene into the red compounds 3,4-didehydrolycopene and torulene. The green peach aphid, Myzus persicae, separated from the pea aphid for ≈45MY also exists as distinct colour variable morphs, with both red and green individuals present. Here, we examined genomic data for both red and green morphs of M. persicae and identified an enlarged (compared to A. pisum) repertoire of 16 carotenoid biosynthetic genes (11 carotenoid desaturases and five carotenoid cyclase/synthase genes). From these, we identify the homolog of A. pisum Tor (here called carotene desaturase 2 or CDE-2) and show through 3D modelling that this homolog can accommodate the torulene precursor lycopene and, through RNA knockdown feeding experiments, demonstrate that disabling CDE-2 expression in red M. persicae clones results in green-coloured offspring. Unlike in A. pisum, we show that functional CDE-2 is present in the genomes of both red and green aphids. However, expression differences between the two colour morphs (350-700 fold CDE-2 overexpression in red clones), potentially driven by variants identified in upstream putative regulatory elements, underpin this phenotype. Thus, whilst aphids have a common origin of their carotenoid biosynthetic pathway, two aphid species separated for over 40MY have evolved very different drivers of intraspecific colour variation.

Antibacterial FANA oligonucleotides as a novel approach for managing the Huanglongbing pathosystem.

Candidatus Liberibacter asiaticus (CLas), a bacterium transmitted by the Asian citrus psyllid, Diaphorina citri, is the causal agent of citrus greening disease, or Huanglongbng (HLB). Currently, vector population suppression with insecticides and tree removal are the most effective strategies for managing the HLB pathosystem. In this study, we assessed the bactericidal capabilities of 2'-deoxy-2'-fluoro-D-arabinonucleic acid antisense oligonucleotides (FANA ASO) both in vitro and in vivo by (1) confirming their capacity to penetrate insect cells, (2) silencing bacterial essential genes, and (3) quantifying reductions in bacterial titer and D. citri transmission. We confirmed that FANA ASO are able to penetrate insect cells without the use of a delivery agent. Expression of an essential gene in the D. citri endosymbiont, Wolbachia (wDi), significantly decreased by 30% following incubation with a wDi-specific FANA ASO. Viability of isolated wDi cells also decreased in response to the FANA ASO treatment. Delivery of a CLas-specific FANA ASO to infected adult D. citri in feeding assays resulted in significant silencing of a CLas essential gene. CLas relative density and transmission were significantly lower among D. citri fed FANA ASO in diet compared to untreated insects. Root infusions of a CLas-specific FANA ASO into infected Citrus trees significantly reduced CLas titer during a 30-day trial. Our results suggest that FANA ASO targeting insect-transmitted plant bacteria or insect endosymbionts may be useful tool for integrated management of agricultural pathogens.

Dual Mechanisms of Action of Self-Delivering, Anti-HIV-1 FANA Oligonucleotides as a Potential New Approach to HIV Therapy.

Currently, the most effective and durable therapeutic option for HIV-1 infection is combination antiretroviral therapy (cART). Although cART is powerful and can delay viral evolution of drug resistance for decades, it is associated with limitations, including an inability to eradicate the virus and a potential for adverse effects. Therefore, it is imperative to discover new HIV therapeutic modalities. In this study, we designed, characterized, and evaluated the in vitro potency of 2'-deoxy-2'-fluoroarabinonucleotide (FANA) modified antisense oligonucleotides (ASOs) targeting highly conserved regions in the HIV-1 genome. Carrier-free cellular internalization of FANA ASOs resulted in strong suppression of HIV-1 replication in HIV-1-infected human primary cells. In vitro mechanistic studies suggested that the inhibitory effect of FANA ASOs can be attributed to RNase H1 activation and steric hindrance of dimerization. Using 5'-RACE PCR and sequencing analysis, we confirmed the presence of human RNase H1-mediated target RNA cleavage products in cells treated with FANA ASOs. We observed no overt cytotoxicity or immune responses upon FANA ASO treatment. Together, our results strongly suggest that FANA ASOs hold great promise for antiretroviral therapy. The dual ability of FANA ASOs to target RNA by recruiting RNase H1 and/or sterically blocking RNA dimerization further enhances their therapeutic potential.

Searching microsatellites in DNA sequences: approaches used and tools developed.

Microsatellite instability associated genomic activities and evolutionary changes have led to a renewed focus on microsatellite research. In last decade, a number of microsatellite mining tools have been introduced based on different computational approaches. The choice is generally made between slow but exhaustive dynamic programming based approaches, or fast and incomplete heuristic methods. Tools based on stochastic approaches are more popular due to their simplicity and added ornamental features. We have performed a comparative evaluation of the relative efficiency of some microsatellite search tools with their default settings. The graphical user interface, the statistical analysis of the output and ability to mine imperfect repeats are the most important criteria in selecting a tool for a particular investigation. However, none of the available tools alone provides complete and accurate information about microsatellites, and a lot depends on the discretion of the user.

Synergistic effects between analogs of DNA and RNA improve the potency of siRNA-mediated gene silencing.

We report that combining a DNA analog (2'F-ANA) with rigid RNA analogs [2'F-RNA and/or locked nucleic acid (LNA)] in siRNA duplexes can produce gene silencing agents with enhanced potency. The favored conformations of these two analogs are different, and combining them in a 1-1 pattern led to reduced affinity, whereas alternating short continuous regions of individual modifications increased affinity relative to an RNA:RNA duplex. Thus, the binding affinity at key regions of the siRNA duplex could be tuned by changing the pattern of incorporation of DNA-like and RNA-like nucleotides. These heavily or fully modified duplexes are active against a range of mRNA targets. Effective patterns of modification were chosen based on screens using two sequences targeting firefly luciferase. We then applied the most effective duplex designs to the knockdown of the eIF4E binding proteins 4E-BP1 and 4E-BP2. We identified modified duplexes with potency comparable to native siRNA. Modified duplexes showed dramatically enhanced stability to serum nucleases, and were characterized by circular dichroism and thermal denaturation studies. Chemical modification significantly reduced the immunostimulatory properties of these siRNAs in human peripheral blood mononuclear cells.

UgMicroSatdb: database for mining microsatellites from unigenes.

Microsatellites, also known as simple sequence repeats (SSRs) or short tandem repeats (STRs), have extensively been exploited as molecular markers for diverse applications. Recently, their role in gene regulation and genome evolution has also been discussed widely. We have developed UgMicroSatdb (Unigene MicroSatellite database), a web-based relational database of microsatellites present in unigene sequences covering 80 genomes. UgMicroSatdb allows microsatellite search using multiple parameters like microsatellite type (simple perfect, compound perfect and imperfect), repeat unit length (mono- to hexa-nucleotide), repeat number, microsatellite length and repeat sequence class. Microsatellites can also be retrieved by specifying EST, cDNA, CDS identity or by using Gene Index, GenBank, UniGene IDs. The database also provides information about trinucleotide repeats encoding various amino acids. Such codon repeats can be searched by specifying characteristics of coded amino acids like charge (basic, acidic or neutral), polarity (polar or non-polar), and their hydrophobic or hydrophilic nature. The nucleotide sequences of the target UniGenes are also provided to facilitate primer designing for PCR amplification of the desired microsatellite. UgMicroSatdb is available at http://ipu.ac.in/usbt/UgMicroSatdb.htm.

EuMicroSatdb: a database for microsatellites in the sequenced genomes of eukaryotes.

BACKGROUND: Microsatellites have immense utility as molecular markers in different fields like genome characterization and mapping, phylogeny and evolutionary biology. Existing microsatellite databases are of limited utility for experimental and computational biologists with regard to their content and information output. EuMicroSatdb (Eukaryotic MicroSatellite database) http://ipu.ac.in/usbt/EuMicroSatdb.htm is a web based relational database for easy and efficient positional mining of microsatellites from sequenced eukaryotic genomes. DESCRIPTION: A user friendly web interface has been developed for microsatellite data retrieval using Active Server Pages (ASP). The backend database codes for data extraction and assembly have been written using Perl based scripts and C++. Precise need based microsatellites data retrieval is possible using different input parameters like microsatellite type (simple perfect or compound perfect), repeat unit length (mono- to hexa-nucleotide), repeat number, microsatellite length and chromosomal location in the genome. Furthermore, information about clustering of different microsatellites in the genome can also be retrieved. Finally, to facilitate primer designing for PCR amplification of any desired microsatellite locus, 200 bp upstream and downstream sequences are provided. CONCLUSION: The database allows easy systematic retrieval of comprehensive information about simple and compound microsatellites, microsatellite clusters and their locus coordinates in 31 sequenced eukaryotic genomes. The information content of the database is useful in different areas of research like gene tagging, genome mapping, population genetics, germplasm characterization and in understanding microsatellite dynamics in eukaryotic genomes.

Biased distribution of microsatellite motifs in the rice genome.

Microsatellites are useful tools to study the extent of divergence between two taxonomic groups that show high sequence similarity. We have compared microsatellite distribution to illustrate genetic variation between the two rice genomes, Oryza sativa L. ssp. indica and Oryza sativa L. ssp. japonica. Microsatellite distribution proved to be non random as certain regions of very high microsatellite density have been identified. Microsatellite density in the subspecies japonica was computed marginally higher than in the subspecies indica in the genomic regions compared between the two subspecies. Unexpectedly high microsatellite densities were observed in 5'-untranslated regions of genes. These regions also displayed a clear motif bias. Some of the longest microsatellite repeats were found in intron sequences. Frequency, as well as motif bias was also noted with respect to the association of microsatellites with transposable elements. Microsatellite mutability values were exemplarily estimated for 90 loci by aligning the microsatellite containing regions between the two genomes. Poor rates of finding an orthologue corresponded with high microsatellite mutability in rice. These insights are likely to play a significant role in selecting microsatellite loci to be used in molecular breeding and studying evolutionary dynamics of the two subspecies.