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As next-generation pathogen detection methods, CRISPR-Cas-based detection methods can perform single-nucleotide polymorphism (SNP) level detection with high sensitivity and good specificity. They do not require any particular equipment, which opens up new possibilities for the accurate detection and identification of Bacillus anthracis. In this study, we developed a complete detection system for B. anthracis based on Cas12a. We used two chromosomally located SNP targets and two plasmid targets to identify B. anthracis with high accuracy. The CR5 target is completely new. The entire detection process can be completed within 90 min without electrical power and with single-copy level sensitivity. We also developed an unaided-eye visualization system based on G4-DNAzyme for use with our CRISPR-Cas12a detection system. This visualization system has good prospects for deployment in field-based point-of-care detection. We used the antisense nucleic acid CatG4R as the detection probe, which showed stronger resistance to interference from components of the solution. CatG4R can also be designed as an RNA molecule for adaptation to Cas13a detection, thereby broadening the scope of the detection system.

Substantial evidence highlighted the critical role of long non-coding RNAs (lncRNA) in driving hepatocarcinogenesis. We hypothesized that functional variants in genome-wide association studies (GWASs) associated loci might alter the expression levels of lncRNAs and contribute to the development of hepatocellular carcinoma (HCC). Here, we prioritized potentially cis-expression quantitative trait loci-based single nucleotide polymorphism (SNP)-lncRNA association together with the physical interaction by the analyses from Hi-C data in GWAS loci of chronic hepatitis B and HCC. Subsequently, by leveraging two-stage case-control study (1738 hepatitis B [HBV]) related HCC cases and 1988 HBV persistent carriers) and biological assays, we identified that rs2647046 was significantly associated with HCC risk (odds ratio = 1.26, 95% CI = 1.11 to 1.43, P = 4.14 × 10-4). Luciferase reporter assays and electrophoretic mobility shift assays showed that rs2647046 A allele significantly increased transcriptional activity via influencing transcript factor binding affinity. Allele-specific chromosome conformation capture assays revealed that enhancer with rs2647046 interacted with the HLA-DQB1-AS1 promoter to allele-specifically influence its expression by CTCF-mediated long-range loop. Cell proliferation assays indicated that HLA-DQB1-AS1 is a potential oncogene in HCC. Our study showed HLA-DQB1-AS1 regulated by a causal SNP in a long-range interaction manner conferred the susceptibility to HCC, suggesting an important mechanism of modulating lncRNA expression for risk-associated SNPs in the etiology of HCC., (© The Author(s) 2021. Published by Oxford University Press. All rights reserved. For Permissions, please email: journals.permissions@oup.com.)

Increasing studies have indicated the critical roles of long non-coding RNAs (lncRNAs) in the tumorigenesis of cancers. LncRNA AGAP2 antisense RNA 1 (AGAP2-AS1) can serve as an oncogenic role in some cancers, including prostate cancer (PCa). However, the underling mechanism of such lncRNA in PCa has not been fully studied. Therefore, it is meaningful to investigate the role and underlying mechanism of AGAP2-AS1 in PCa. AGAP2-AS1 was confirmed to be highly expressed in PCa cells. Functionally, AGAP2-AS1 silencing inhibited cell proliferation, migration, invasion and epithelial-mesenchymal transition process and induced apoptosis. According to mechanism assays, AGAP2-AS1 sponged miR-628-5p, which was found to restrain PCa cell growth. Besides, FOXP2 was identified as a target gene of miR-628-5p, and its expression was negatively regulated by miR-628-5p and positively modulated by AGAP2-AS1. Importantly, we found that FOXP2 could function as the upstream gene of AGAP2-AS1. Through rescue experiments, we discovered that FOXP2 up-regulation countered AGAP2-AS1 knockdown-mediated inhibition on PCa cell growth. Finally, it was found that AGAP2-AS1 could activate WNT pathway, and LiCl could reverse the influence of AGAP2-AS1 on PCa biological behaviors. To conclude, AGAP2-AS1/miR-628-5p/FOXP2 feedback loop facilitated PCa cell growth via activating WNT pathway., (© The Author(s) 2021. Published by Oxford University Press. All rights reserved. For Permissions, please email: journals.permissions@oup.com.)

The blood system serves as a key model for cell differentiation and cancer. It is orchestrated by precise spatiotemporal expression of crucial transcription factors. One of the key master regulators in the hematopoietic systems is PU.1. Reduced levels of PU.1 are characteristic for human acute myeloid leukemia (AML) and are known to induce AML in mouse models. Here, we show that transcriptional downregulation of PU.1 is an active process involving an alternative promoter in intron 3 that is induced by RUNX transcription factors driving noncoding antisense transcription. Core-binding factor (CBF) fusions RUNX1-ETO and CBFβ-MYH11 in t(8;21) and inv(16) AML, respectively, activate the PU.1 antisense promoter that results in a shift from sense toward antisense transcription and myeloid differentiation blockade. In patients with CBF-AML, we found that an elevated antisense/sense transcript and promoter accessibility ratio represents a hallmark compared with normal karyotype AML or healthy CD34+ cells. Competitive interaction of an enhancer with the proximal or the antisense promoter forms a binary on/off switch for either myeloid or T-cell development. Leukemic CBF fusions thus use a physiological mechanism used by T cells to decrease sense transcription. Our study is the first example of a sense/antisense promoter competition as a crucial functional switch for gene expression perturbation by oncogenes. Hence, this disease mechanism reveals a previously unknown Achilles heel for future precise therapeutic targeting of oncogene-induced chromatin remodeling., (© 2021 by The American Society of Hematology.)

Brain-derived neurotrophic factor-antisense (BDNF-AS) is a long non-coding RNA with tens of alternatively spliced variants being transcribed from 11p14 cytogenetic band. As a naturally occurring anti-sense, it regulates expression of BDNF, a factor which as essential roles in the pathoetiology of neurodevelopmental diseases. Notably, BDNF-AS has been reported to be down-regulated in colorectal cancer, osteosarcoma, esophageal cancer, glioblastoma, prostate cancer, cervical cancer and breast cancer. This lncRNA has direct/indirect functional interactions with GSK-3β, EZH2, miR-214, PABPC1, RAX2, DLG5, p53 and ADAR as well as RNH1/TRIM21/mTOR signaling. In prostate and breast cancers, down-regulation of BDNF-AS has been associated with poor clinical outcome. In the present review, we assessed the existing literature on the role of BDNF-AS in this process and summarized the available data in three distinct sections based of the methodology of experiments and source of expression assays. We also summarized the role of BDNF-AS in non-neoplastic conditions., (Copyright © 2021 The Authors. Published by Elsevier Masson SAS.. All rights reserved.)

Nanobodies (VHHs) are the single variable immunoglobulin domains of heavy chain antibodies (hcAbs) that naturally occur in alpacas and other camelids. The two variable domains of conventional antibodies typically interact via a hydrophobic interface. In contrast, the corresponding surface area of nanobodies is hydrophilic, rendering these single immunoglobulin domains highly soluble, robust to harsh environments, and exceptionally easy to format into bispecific reagents. In homage to Geoffrey Burnstock, the pioneer of purinergic signaling, we provide a brief history of nanobody-mediated modulation of purinergic signaling, using our nanobodies targeting P2X7 and the NAD + -metabolizing ecto-enzymes CD38 and ARTC2.2 as examples., (Copyright © 2020 Elsevier Inc. All rights reserved.)

Tyrosine kinase inhibitor is an effective chemo-therapeutic drug against tumors with deregulated EGFR pathway. Recently, a genetic variant rs10251977 (G>A) in exon 20 of EGFR reported to act as a prognostic marker for HNSCC. Genotyping of this polymorphism in oral cancer patients showed a similar frequency in cases and controls. EGFR-AS1 expressed significantly high level in tumors and EGFR-A isoform expression showed significant positive correlation (r = 0.6464, p < 0.0001) with reference to EGFR-AS1 expression levels, consistent with larger TCGA HNSCC tumor dataset. Our bioinformatic analysis showed enrichment of alternative splicing marks H3K36me3 and presence of intronic polyA sites spanning around exon 15a and 15b of EGFR facilitates skipping of exon 15b, thereby promoting the splicing of EGFR-A isoform. In addition, high level expression of PTBP1 and its binding site in EGFR and EGFR-AS1 enhances the expression of EGFR-A isoform (r = 0.7404, p < 0.0001) suggesting that EGFR-AS1 expression modulates the EGFR-A and D isoforms through alternative splicing. In addition, this polymorphism creates a binding site for miR-891b in EGFR-AS1 and may negatively regulate the EGFR-A. Collectively, our results suggested the presence of genetic variant in EGFR-AS1 modulates the expression of EGFR-D and A isoforms.

In situ hybridization (ISH) and immunohistochemistry (IHC) are essential tools to characterize SARS-CoV-2 infection and tropism in naturally and experimentally infected animals and also for diagnostic purposes. Here, we describe three RNAscope ® -based ISH assays targeting the ORF1ab, spike, and nucleocapsid genes and IHC assays targeting the spike and nucleocapsid proteins of SARS-CoV-2.

GGGGCC hexanucleotide repeat expansions (HREs) in C9orf72 cause amyotrophic lateral sclerosis (ALS) and frontotemporal dementia (FTD) and lead to the production of aggregating dipeptide repeat proteins (DPRs) via repeat associated non-AUG (RAN) translation. Here, we show the similar intronic GGCCTG HREs that causes spinocerebellar ataxia type 36 (SCA36) is also translated into DPRs, including poly(GP) and poly(PR). We demonstrate that poly(GP) is more abundant in SCA36 compared to c9ALS/FTD patient tissue due to canonical AUG-mediated translation from intron-retained GGCCTG repeat RNAs. However, the frequency of the antisense RAN translation product poly(PR) is comparable between c9ALS/FTD and SCA36 patient samples. Interestingly, in SCA36 patient tissue, poly(GP) exists as a soluble species, and no TDP-43 pathology is present. We show that aggregate-prone chimeric DPR (cDPR) species underlie the divergent DPR pathology between c9ALS/FTD and SCA36. These findings reveal key differences in translation, solubility, and protein aggregation of DPRs between c9ALS/FTD and SCA36., Competing Interests: Declaration of Interests The authors declare no competing interests., (Copyright © 2020 Elsevier Inc. All rights reserved.)

The fem-1 gene in Caenorhabditis elegans is involved in sex differentiation; it is specifically required for all aspects of male development. In this study, the full-length cDNA of the fem-1 (Pvfem-1) gene was isolated from the Pacific whiteleg shrimp Penaeus vannamei. The Pvfem-1 transcript is 3778 nt long and encodes a putative protein (PvFEM-1) of 638 amino acids that presented eight ankyrin repeats. The translated protein showed a significant (P < 0.05) structural similitude by superposition with C. elegans FEM-1 protein. Pvfem-1 expression was evaluated by qPCR and in situ hybridization (ISH) during embryogenesis, larval development, and gonads of both genders in subadult and adult life stages. Pvfem-1 was found expressed in brain, intestine, hepatopancreas, and in the gonads of both genders in subadults and adults when quantified by RT-qPCR. A significant finding was the discovery of a natural antisense transcript (NAT) of Pvfem-1 by ISH. It was present in the oocyte nucleus of subadult female shrimp gonads but was not seen within oocytes from adult females, although it was detected in follicular cells, suggesting a possible post-transcriptional regulation of Pvfem-1 in female gonad. Conversely, in males, no NAT was observed, and Pvfem-1 was found expressed in spermatogonia of both, subadult and adult shrimps indicating a function in male sexual differentiation and gametes generation. This study represents the first step for future functional analysis that is expected to contribute to clarifying the role of Pvfem-1 in sex differentiation and determination., (Copyright © 2019 Elsevier B.V. All rights reserved.)

Natural antisense transcripts (NATs) have recently been associated with the development of human cancers. Recent studies have shown that a natural antisense transcript (NAT) is present in Sirt1 gene which encodes a NAD-dependent deacetylase. Interestingly, expression of Sirt1 mRNA changes during development and progression of human cancers. However, it remains unclear to what extent Sirt1 antisense transcript (AS) may contribute to changes in the expression of Sirt1 mRNA. To determine this, we used quantitative measurement of RNA to reveal relationship between Sirt1 mRNA and Sirt1-AS across human cancer tissues, cell lines and stem cells. While Sirt1 mRNA level was increased in cancer cell lines and cancer tissues, the expression level of Sirt1-AS was lower in cancers compared to controls. This inverse correlation was observed in the expression of Sirt1 sense and antisense transcripts in normal and cancer tissues suggesting a functional role for Sirt1-AS in regulation of Sirt1 mRNA.

Background: Drug-mediated disruption of IL17A, IL17F and IL17RA proteins is effective in psoriasis. However, disruption of the IL17 pathway by functional mutations has so far only been shown to affect risk in IL23R and TRAF3IP2. It is unclear whether this is due to rarity of disruptive mutations., Objective: (a) To delineate the prevalence of mutations in key IL17 pathway genes and (b) to identify candidate regulatory sites acting on IL23R, IL17A, IL17RA and TRAF3IP2 from a distance., Methods: Extraction of mutation frequencies from ExAc data, evolutionary sequence alignment; mapping of long-range interacting (LRI) enhancers; and genetic association testing in a novel psoriasis cohort., Results: The prevalence of disruptive mutations in genes such as IL17RA is sufficient to have been detectable by existing data sets. Therefore, lack of their association with psoriasis indicates that genetic risk primarily resides in variants acting from a distance. We identify two LRI enhancer sites, regulating IL17RA and TRAF3IP2, respectively. The TRAF3IP2 regulator localizes to the TRAF3IP2 antisense promoter, suggesting feedback regulation. Both LRI sites are associated with psoriasis in a novel Scottish psoriasis cohort and the TRAF3IP2-LRI at rs71562294 replicates in the WTCCC cohort., Conclusion: Genetic risk for psoriasis may be encoded at LRI sites regulating IL17 pathway genes from a distance., (© 2018 John Wiley & Sons A/S. Published by John Wiley & Sons Ltd.)

For patients with osteoporosis, the inability of osteogenic differentiation is the key reason for bone loss. In this study, we investigated the expression and function of long non-coding RNA BDNF-AS in mesenchymal stem cell-derived osteogenic differentiation. Mouse bone marrow-derived mesenchymal stem cells (BMMSCs) were cultured in vitro and induced toward osteogenic differentiation. Quantitative real-time PCR (qRT-PCR) was used to evaluate gene expressions of BDNF-AS and BDNF during osteogenic differentiation. BMMSCs were also extracted from ovariectomized (OVX) mice. The dynamic change of BDNF-AS in OVX-derived BMMSCs during osteogenic differentiation was also evaluated. Lentivirus was used to upregulate BDNF-AS in BMMSCs. The effects of BDNF-AS upregulation on BMMSCs' proliferation and osteogenic differentiation were then evaluated. In addition, qRT-PCR and western blot were applied to further examine the effect of BDNF-AS upregulation on osteogenesis-associated signaling pathways, including BDNF, OPN, and Runx2, in osteogenic differentiation. BDNF-AS was downregulated, whereas BDNF was upregulated in osteogenic differentiation of BMMSCs. Among OVX-derived BMMSCs, BDNF-AS expression was upregulated during osteogenic differentiation. Lentivirus-induced BDNF-AS upregulation promoted BMMSCs self-proliferation but inhibited osteogenic differentiation, as demonstrated by proliferation, alizarin red staining, and alkaline phosphatase activity assays, respectively. QRT-PCR and western blot demonstrated that BDNF, OPN, and Runx2 were downregulated by BDNF-AS upregulation in the differentiated BMMSCs. BDNF-AS is dynamically regulated in osteogenic differentiation. Upregulating BDNF-AS inhibits osteogenesis, possibly through inverse regulation on BDNF and osteogenic signaling pathways.

Age-related macular degeneration (AMD) predominantly affects the retina and retinal pigment epithelium in the posterior eye. While there are numerous studies investigating the non-coding transcriptome of retina and RPE, few significant differences between AMD and normal tissues have been reported. Strand specific RNA sequencing of both peripheral retina (PR) and RPE-Choroid-Sclera (PRCS), in both AMD and matched normal controls were generated. The transcriptome analysis reveals a highly significant and consistent impact on anti-sense transcription as well as moderate changes in the regulation of non-coding (sense) RNA. Hundreds of genes that do not express anti-sense transcripts in normal PR and PRCS demonstrate significant anti-sense expression in AMD in all patient samples. Several pathways are highly enriched in the upregulated anti-sense transcripts-in particular the EIF2 signaling pathway. These results call for a deeper exploration into anti-sense and noncoding RNA regulation in AMD and their potential as therapeutic targets.

Sense and antisense peptides, i.e. peptides specified by complementary DNA and RNA sequences, interact with increased probability. Biro, Blalock, Mekler, Root-Bernstein and Siemion investigated the recognition rules of peptide-peptide interaction based on the complementary coding of DNA and RNA sequences in 3'→5' and 5'→3' directions. After more than three decades of theoretical and experimental investigations, the efficiency of this approach to predict peptide-peptide binding has been experimentally verified for more than 50 ligand-receptor systems, and represents a promising field of research. The natural genetic coding algorithm for sense and antisense peptide interactions combines following elements: of amino acid physico-chemical properties, stereochemical interaction, and bidirectional transcription. The interplay of these factors influences the specificity of sense-antisense peptide interactions, and affects the selection and evolution of peptide ligand-receptor systems. Complementary mRNA codon-tRNA anticodon complexes, and recently discovered Carter-Wolfenden tRNA acceptor-stem code, provide the basis for the rational modeling of peptide interactions based on their hydrophobic and lipophilic amino acid physico-chemical properties. It is shown that the interactions of complementary amino acid pairs according to the hydrophobic and lipophilic properties strongly depend on the central (second) purine base of the mRNA codon and its pyrimidine complement of the tRNA anticodon. This enables the development of new algorithms for the analysis of structure, function and evolution of protein and nucleotide sequences that take into account the residue's tendency to leave water and enter a nonpolar condensed phase considering its mass, size and accessible surface area. The practical applications of the sense-antisense peptide modeling are illustrated using different interaction assay types based on: microscale thermophoresis (MST), tryptophan fluorescence spectroscopy (TFS), nuclear magnetic resonance spectroscopy (NMR), and magnetic particles enzyme immunoassay (MPEIA). Various binding events and circumstances were considered, e.g., in situations with-short antisense peptide ligand (MST), L- and D-enantiomer acceptors (TFS), in low affinity conditions (NMR), and with more than one antisense peptide targeting hormone (MPEIA)., (Copyright © 2017 The Authors. Published by Elsevier B.V. All rights reserved.)

Alu elements in the human genome are present in more than one million copies, accounting for 10% of the genome. However, the biological functions of most Alu repeats are unknown. In this present study, we detected the effects of Alu elements on EGFP gene expression using a plasmid system to find the roles of Alu elements in human genome. We inserted 5'-4TMI-Alus-CMV promoter-4TMI-Alus (or antisense Alus)-3' sequences into the pEGFP-C1 vector to construct expression vectors. We altered the copy number of Alus, the orientation of the Alus, and the presence of an enhancer (4TMI) in the inserted 5'-4TMI-Alus-CMV promoter-4TMI-Alus (or antisense Alus)-3' sequences. These expression vectors were stably transfected into HeLa cells, and EGFP reporter gene expression was determined. Our results showed that combined sense-antisense Alu elements activated the EGFP reporter gene in the presence of enhancers and stable transfection. The combined sense-antisense Alu vectors carrying four copies of Alus downstream of inserted CMV induced much stronger EGFP gene expression than two copies. Alus downstream of inserted CMV were replaced to AluJBs (having 76% homology with Alu) to construct expression vectors. We found that combined sense-antisense Alu (or antisense AluJB) vectors induced strong EGFP gene expression after stable transfection and heat shock. To further explore combined sense-antisense Alus activating EGFP gene expression, we constructed Tet-on system vectors, mini-C1-Alu-sense-sense and mini-C1-Alu-sense-antisense (EGFP gene was driven by mini-CMV). We found that combined sense-antisense Alus activated EGFP gene in the presence of reverse tetracycline repressor (rTetR) and doxycycline (Dox). Clone experiments showed that Mini-C1-Alu-sense-antisense vector had more positive cells than that of Mini-C1-Alu-sense-sense vector. The results in this paper proved that Alu repetitive sequences inhibited gene expression and combined sense-antisense Alus activated EGFP reporter gene when Alu transcribes, which suggests that Alus play roles in maintaining gene expression (silencing genes or activating genes) in human genome.

Antisense transcription is a prevalent feature at mammalian promoters. Previous studies have primarily focused on antisense transcription initiating upstream of genes. Here, we characterize promoter-proximal antisense transcription downstream of gene transcription starts sites in human breast cancer cells, investigating the genomic context of downstream antisense transcription. We find extensive correlations between antisense transcription and features associated with the chromatin environment at gene promoters. Antisense transcription downstream of promoters is widespread, with antisense transcription initiation observed within 2 kb of 28% of gene transcription start sites. Antisense transcription initiates between nucleosomes regularly positioned downstream of these promoters. The nucleosomes between gene and downstream antisense transcription start sites carry histone modifications associated with active promoters, such as H3K4me3 and H3K27ac. This region is bound by chromatin remodeling and histone modifying complexes including SWI/SNF subunits and HDACs, suggesting that antisense transcription or resulting RNA transcripts contribute to the creation and maintenance of a promoter-associated chromatin environment. Downstream antisense transcription overlays additional regulatory features, such as transcription factor binding, DNA accessibility, and the downstream edge of promoter-associated CpG islands. These features suggest an important role for antisense transcription in the regulation of gene expression and the maintenance of a promoter-associated chromatin environment.

NuA4 histone lysine (K) acetyltransferase (KAT) promotes transcriptional initiation of TATA-binding protein (TBP)-associated factor (TAF)-dependent ribosomal protein genes. TAFs have also been recently found to enhance antisense transcription from the 3' end of the GAL10 coding sequence. However, it remains unknown whether, like sense transcription of the ribosomal protein genes, TAF-dependent antisense transcription of GAL10 also requires NuA4 KAT. Here, we show that NuA4 KAT associates with the GAL10 antisense transcription initiation site at the 3' end of the coding sequence. Such association of NuA4 KAT depends on the Reb1p-binding site that recruits Reb1p activator to the GAL10 antisense transcription initiation site. Targeted recruitment of NuA4 KAT to the GAL10 antisense transcription initiation site promotes GAL10 antisense transcription. Like NuA4 KAT, histone H3 K4/36 methyltransferases and histone H2B ubiquitin conjugase facilitate GAL10 antisense transcription, while the Swi/Snf and SAGA chromatin remodeling/modification factors are dispensable for antisense, but not sense, transcription of GAL10. Taken together, our results demonstrate for the first time the roles of NuA4 KAT and other chromatin regulatory factors in controlling antisense transcription, thus illuminating chromatin regulation of antisense transcription., (Copyright © 2016, American Society for Microbiology. All Rights Reserved.)

Glioblastoma (GBM) generally exhibits high IC50 values for its standard drug treatment, temozolomide (TMZ). MicroRNA-21 (miR-21) is an oncomiR overexpressed in GBM, thus controlling important aspects of glioma biology. We hypothesized that PLGA nanoparticles carrying antisense miR-21 to glioblastoma cells might beneficially knock down endogenous miR-21 prior to TMZ treatment. PLGA nanoparticles encapsulating antisense miR-21 were effective in intracellular delivery and sustained silencing (p < 0.01) of miR-21 function in U87 MG, LN229, and T98G cells. Prior antisense miR-21 delivery significantly reduced the number of viable cells (p < 0.001), and increased (1.6-fold) cell cycle arrest at G2/M phase upon TMZ treatment in U87 MG cells. There was overexpression of the miR-21 target genes PTEN (by 67%) and caspase-3 (by 15%) upon cotreatment. This promising PLGA nanoparticle-based platform for antisense miR-21 delivery to GBM is an effective cotherapeutic strategy in cell culture, warranting the need for further studies prior to future clinical translation.

Heart failure (HF) is the end result of a diverse set of causes such as genetic cardiomyopathies, coronary artery disease, and hypertension and represents the primary cause of hospitalization in Europe. This serious clinical disorder is mostly associated with pathological remodeling of the myocardium, pump failure, and sudden death. While the survival of HF patients can be prolonged with conventional pharmacological therapies, the prognosis remains poor. New therapeutic modalities are thus needed that will target the underlying causes and not only the symptoms of the disease. Under chronic cardiac stress, small noncoding RNAs, in particular microRNAs, act as critical regulators of cardiac tissue remodeling and represent a new class of therapeutic targets in patients suffering from HF. Here, we focus on the potential use of microRNA inhibitors as a new treatment paradigm for HF.

Background: Long noncoding RNAs (IncRNAs) have been demonstrated to be associated with human cancer. However, the clinical value of most lncRNAs to serve as the biomarkers for gastric cancer remains largely unknown. In this study, we examined the relationship between the expression levels of IncRNA, PCNA antisense RNA 1 (PCNA-AS1), and clinicopathological characteristics of gastric cancer., Methods: A total of 90 tissue samples from patients with gastric cancer were collected, and the IncRNA PCNA-AS1 levels in cancer and paired non-cancer tissues was detected by real-time quantitative RT-PCR (qRT-PCR). The relationship between PCNA-AS1 levels and the clinicopathological characteristics were then evaluated., Results: PCNA-AS1 expression levels were increased in 68 of 90 gastric cancer tissues (75.56%) and were associated with invasion (p = 0.038), but not associated with other clinicopathological characteristics. The expression levels of PCNA-AS1 were also related with immunohistochemical biomarkers of BRCA1 (p = 0.043) and RRM1 (p = 0.023)., Conclusions: These data indicated that IncRNA-PCNA-AS1 may participate in the gastric cancer carcinogenesis and development and may serve as a new biomarker for patients with gastric cancer.

Expanded GGGGCC (G4C2) nucleotide repeats within the C9ORF72 gene are the most common genetic mutation associated with both amyotrophic lateral sclerosis (ALS) and frontotemporal dementia (FTD). Sense and antisense transcripts of these expansions are translated to form five dipeptide repeat proteins (DRPs). We employed primary cortical and motor neuron cultures, live-cell imaging, and transgenic fly models and found that the arginine-rich dipeptides, in particular Proline-Arginine (PR), are potently neurotoxic. Factors that anticipated their neurotoxicity included aggregation in nucleoli, decreased number of processing bodies, and stress granule formation, implying global translational dysregulation as path accountable for toxicity. Nuclear PR aggregates were also found in human induced motor neurons and postmortem spinal cord tissues from C9ORF72 ALS and ALS/FTD patients. Intronic G4C2 transcripts, but not loss of C9ORF72 protein, are also toxic to motor and cortical neurons. Interestingly, G4C2 transcript-mediated neurotoxicity synergizes with that of PR aggregates, suggesting convergence of mechanisms., (Copyright © 2014 Elsevier Inc. All rights reserved.)

While transforming growth factor-β1 (TGF-β1) can regulate odontoblast differentiation in tooth crown morphogenesis, its effects on cells including stem cells from the apical papilla (SCAPs) involved in root formation are unclear. Nuclear factor I-C (NFIC) has been implicated in the regulation of root development, and interplay with TGF-β1 signaling has been reported in some cell types. We hypothesize that NFIC and TGF-β1 are important to the behavior of SCAPs and that the interplay between these molecules controls the regulation of the odontogenic differentiation of SCAPs. TGF-β1 inhibited the proliferation of SCAPs and their mineralization. Real-time polymerase chain-reaction (RT-PCR) and Western blot results showed that TGF-β1 significantly decreased osteogenic/dentinogenic gene expression. The inhibition of TGF-β/Smad signaling (SIS3) attenuated the suppressive effect of TGF-β1 on SCAPs. Importantly, overexpression of NFIC antagonized the effects of TGF-β1 on SCAPs, while knockdown of NFIC enhanced these effects, demonstrating a key regulatory role for NFIC in modulating TGF-β1 signaling in SCAPs. We conclude that this interplay between NFIC and TGF-β1 regulates SCAPs behavior and can determine the differentiation of these cells. These signaling interactions help inform the development of regenerative strategies aimed at root growth and development in immature teeth for endodontic treatment.

DNA:RNA hybrid formation is emerging as a significant cause of genome instability in biological systems ranging from bacteria to mammals. Here we describe the genome-wide distribution of DNA:RNA hybrid prone loci in Saccharomyces cerevisiae by DNA:RNA immunoprecipitation (DRIP) followed by hybridization on tiling microarray. These profiles show that DNA:RNA hybrids preferentially accumulated at rDNA, Ty1 and Ty2 transposons, telomeric repeat regions and a subset of open reading frames (ORFs). The latter are generally highly transcribed and have high GC content. Interestingly, significant DNA:RNA hybrid enrichment was also detected at genes associated with antisense transcripts. The expression of antisense-associated genes was also significantly altered upon overexpression of RNase H, which degrades the RNA in hybrids. Finally, we uncover mutant-specific differences in the DRIP profiles of a Sen1 helicase mutant, RNase H deletion mutant and Hpr1 THO complex mutant compared to wild type, suggesting different roles for these proteins in DNA:RNA hybrid biology. Our profiles of DNA:RNA hybrid prone loci provide a resource for understanding the properties of hybrid-forming regions in vivo, extend our knowledge of hybrid-mitigating enzymes, and contribute to models of antisense-mediated gene regulation. A summary of this paper was presented at the 26th International Conference on Yeast Genetics and Molecular Biology, August 2013.

White spot syndrome caused by white spot syndrome virus (WSSV) is one of the most threatening diseases of shrimp culture industry. Previous studies have successfully demonstrated the use of DNA- and RNA-based vaccines to protect WSSV infection in shrimp. In the present study, we have explored the protective efficacy of antisense constructs directed against WSSV proteins, VP24, and VP28, thymidylate synthase (TS), and ribonucleotide reductase-2 (RR2) under the control of endogenous shrimp histone-3 (H3) or penaedin (Pn) promoter. Several antisense constructs were generated by inserting VP24 (pH3-VP24, pPn-VP24), VP28 (pH3-VP28, pPn-VP28), TS (pH3-TS, pPn-TS), and RR2 (pH3-RR2) in antisense orientation. These constructs were tested for their protective potential in WSSV infected cell cultures, and their effect on reduction of the viral load was assessed. A robust reduction in WSSV copy number was observed upon transfection of antisense constructs in hemocyte cultures derived from Penaeus monodon and Scylla serrata. When tested in vivo, antisense constructs offered a strong protection in WSSV challenged P. monodon. Constructs expressing antisense VP24 and VP28 provided the best protection (up to 90 % survivability) with a corresponding decrease in the viral load. Our work demonstrates that shrimp treated with antisense constructs present an efficient control strategy for combating WSSV infection in shrimp aquaculture.

The rearrangement of pre-existing genes has long been thought of as the major mode of new gene generation. Recently, de novo gene birth from non-genic DNA was found to be an alternative mechanism to generate novel protein-coding genes. However, its functional role in human disease remains largely unknown. Here we show that NCYM, a cis-antisense gene of the MYCN oncogene, initially thought to be a large non-coding RNA, encodes a de novo evolved protein regulating the pathogenesis of human cancers, particularly neuroblastoma. The NCYM gene is evolutionally conserved only in the taxonomic group containing humans and chimpanzees. In primary human neuroblastomas, NCYM is 100% co-amplified and co-expressed with MYCN, and NCYM mRNA expression is associated with poor clinical outcome. MYCN directly transactivates both NCYM and MYCN mRNA, whereas NCYM stabilizes MYCN protein by inhibiting the activity of GSK3β, a kinase that promotes MYCN degradation. In contrast to MYCN transgenic mice, neuroblastomas in MYCN/NCYM double transgenic mice were frequently accompanied by distant metastases, behavior reminiscent of human neuroblastomas with MYCN amplification. The NCYM protein also interacts with GSK3β, thereby stabilizing the MYCN protein in the tumors of the MYCN/NCYM double transgenic mice. Thus, these results suggest that GSK3β inhibition by NCYM stabilizes the MYCN protein both in vitro and in vivo. Furthermore, the survival of MYCN transgenic mice bearing neuroblastoma was improved by treatment with NVP-BEZ235, a dual PI3K/mTOR inhibitor shown to destabilize MYCN via GSK3β activation. In contrast, tumors caused in MYCN/NCYM double transgenic mice showed chemo-resistance to the drug. Collectively, our results show that NCYM is the first de novo evolved protein known to act as an oncopromoting factor in human cancer, and suggest that de novo evolved proteins may functionally characterize human disease.

Plasmodium vivax is the most geographically widespread human malaria parasite causing approximately 130-435 million infections annually. It is an economic burden in many parts of the world and poses a public health challenge along with the other Plasmodium sp. The biology of this parasite is less studied and poorly understood, in spite of these facts. Emerging evidence of severe complications due to infections by this parasite provides an impetus to focus research on the same. Investigating the parasite directly from infected patients is the best way to study its biology and pathogenic mechanisms. Gene expression studies of this parasite directly obtained from the patients has provided evidence of gene regulation resulting in varying amount of transcript levels in the different blood stages. The mechanisms regulating gene expression in malaria parasites are not well understood. Discovery of Natural Antisense Transcripts (NATs) in Plasmodium falciparum has suggested that these might play an important role in regulating gene expression. We report here the genome-wide occurrence of NATs in P. vivax parasites from patients with differing clinical symptoms. A total of 1348 NATs against annotated gene loci have been detected using a custom designed microarray with strand specific probes. Majority of NATs identified from this study shows positive correlation with the expression pattern of the sense (S) transcript. Our data also shows condition specific expression patterns of varying S and antisense (AS) transcript levels. Genes with AS transcripts enrich to various biological processes. To our knowledge this is the first report on the presence of NATs from P. vivax obtained from infected patients with different disease complications. The data suggests differential regulation of gene expression in diverse clinical conditions, as shown by differing sense/antisense ratios and would lead to future detailed investigations of gene regulation., (Copyright © 2013 Elsevier B.V. All rights reserved.)

HOTAIR (HOX antisense intergenic RNA) is a long noncoding RNA (lncRNA) that is transcribed from the antisense strand of homeobox C gene locus in chromosome 12. HOTAIR coordinates with chromatin-modifying enzymes and regulates gene silencing. It is overexpressed in various carcinomas including breast cancer. Herein, we demonstrated that HOTAIR is crucial for cell growth and viability and its knockdown induced apoptosis in breast cancer cells. We also demonstrated that HOTAIR is transcriptionally induced by estradiol (E2). Its promoter contains multiple functional estrogen response elements (EREs). Estrogen receptors (ERs) along with various ER coregulators such as histone methylases MLL1 (mixed lineage leukemia 1) and MLL3 and CREB-binding protein/p300 bind to the promoter of HOTAIR in an E2-dependent manner. Level of histone H3 lysine-4 trimethylation, histone acetylation, and RNA polymerase II recruitment is enriched at the HOTAIR promoter in the presence of E2. Knockdown of ERs and MLLs downregulated the E2-induced HOTAIR expression. Thus, similar to protein-coding gene transcription, E2-induced transcription of antisense transcript HOTAIR is coordinated via ERs and ER coregulators, and this mechanism of HOTAIR overexpression potentially contributes towards breast cancer progression., (Published by Elsevier Ltd.)

RepB is the pMV158-encoded protein that initiates rolling-circle replication of this promiscuous plasmid. Availability of RepB is rate-limiting for the plasmid replication process, and therefore the repB gene encoding the protein is subjected to strict control. Two trans-acting plasmid elements, CopG and the antisense RNAII, are involved in controlling the synthesis of the initiator at the transcriptional and translational level, respectively. In addition to this dual control of repB expression that senses and corrects fluctuations in plasmid copy number, proper availability of RepB also relies on the adequate functionality of the transcription and translation initiation regulatory signals. Translation of repB has been postulated to depend on an atypical ribosome binding site that precedes its start codon, although such a hypothesis has never been proved. To define sequences involved in translation of repB, several mutations in the translation initiation region of the repB mRNA have been characterized by using an Escherichia coli in vitro expression system wherein the synthesis of RepB was detected and quantified. We showed that translation of repB is not coupled to that of copG and depends only on its own initiation signals. The atypical ribosome binding site, as it was defined, is not involved in translation initiation. However, the sequence just upstream of the repB start codon, encompassing the proximal box of the atypical ribosome binding site and the four bases immediately downstream of it, is indeed important for efficient translation of repB. The high degree of conservation of this sequence among the rep genes of plasmids of the same pMV158 family supports its relevancy as a translation initiation signal in mRNAs without a recognizable Shine-Dalgarno sequence., (Copyright © 2013 Elsevier Inc. All rights reserved.)

Objective: To study the effects of antisense p38α mitogen-activated protein kinase (hereinafter referred to as p38α) on myocardial cells exposed to hypoxia and burn serum., Methods: Thirty adult SD rats were inflicted with 40% TBSA full-thickness burn on the back to obtain burn serum. The myocardial cells were isolated from 80 neonatal SD rats and cultured, then they were divided into 4 groups according to the random number table: normal control group (N, ordinary culture without any treatment), hypoxia+burn serum group (HB, exposed to hypoxia after being treated with 10% burn rat serum), hypoxia+burn serum+infection group (HBI, exposed to hypoxia and 10% burn rat serum after being infected with antisense p38α gene-carrying adenovirus), hypoxia+burn serum+empty vector infection group (exposed to hypoxia and 10% burn rat serum after being infected with adenovirus empty vector). At post hypoxia hour (PHH) 1, 3, 6, and 12, mRNA and protein expression levels of p38α in the latter 3 groups were determined by RT-PCR and Western blotting, cell viability was determined by methylthianolyldiphenyl-tetrazolium bromide assay, and lactate dehydrogenase (LDH) activity was assayed at the same time point. At PHH 1, 6, and 12, apoptosis rate of myocardial cells was assessed by annexin V staining method. The indexes of group N were determined with the methods mentioned-above. Three wells were set at each time point in each group. Data were processed with one-way analysis of variance and LSD- t test., Results: (1) At PHH 1, 3, and 6, the p38α mRNA level was higher in group HB than in group N and group HBI (with t values from 2.725 to 4.375, P values all below 0.05). (2) At PHH 1, 3, and 6, the p38α protein level was higher in group HB than those in group N and group HBI (with t values from 5.351 to 7.981, P values all below 0.01). (3) At PHH 3, 6, and 12, the cell viability in group HB (0.115 ± 0.007, 0.104 ± 0.006, 0.094 ± 0.005) was lower than that in group N (0.141 ± 0.014) and group HBI (0.136 ± 0.009, 0.124 ± 0.010, 0.112 ± 0.007, with t values from 2.357 to 6.812, P values all below 0.05). (4) The LDH activity was up-regulated in group HB as compared with that in group N and group HBI at each time point (with t values from 22.753 to 201.273, P values all below 0.01). (5) At PHH 1, 6, and 12, the apoptosis rate of myocardial cells in group HB [(5.4 ± 0.7)%, (8.7 ± 1.1)%, (13.6 ± 1.7)%] was higher than that of group N [(3.1 ± 0.3)%] and group HBI [(4.3 ± 0.5)%, (5.1 ± 0.7)%, (7.2 ± 0.9)%, with t values from 2.345 to 9.700, P < 0.05 or P < 0.01]., Conclusions: Antisense p38α can protect the myocardial cells from the injury of hypoxia and burn serum.

Objective: To explore the effects of the magnetic c-erbB-2 antisense probe of different concentrations on the morphology and expression of SK-Br-3 cancer cells in vitro., Methods: Breast cancer SK-Br-3 cells were transfected for 24 h by antisense probe at an iron concentration of 5, 10, 25, 50, and 100 mg/L, respectively. The distribution and content of iron particles in SK-Br-3 cells was determined by Prussian blue staining, electron microscopy, and atomic absorption spectrometry. Cell viability was observed by trypan-blue exclusion and CCK-8 test. The protein expression of c-erbB-2 was assessed by the Western blot analysis. The changes of the signal strength were considered by magnetic resonance imaging (MRI)., Results: c-erbB-2 antisense probe was uptake by SK-Br-3 cells in a concentration-dependence manner within a certain range (5, 10, and the Medicine Scientific Research Project of Chongqing Health Bureau (062025)25 mg/L). When the probe concentration was 25 mg/L, iron content in cells was (18.38±0.28) pg, the cell vitality, survival, and c-erbB-2 protein expression were reduced significantly (all P<0.05), and the T2 value was lower significantly (P<0.05). However, the results of 50 mg/L or 100 mg/L group showed no significant difference with the 25 mg/L group (P>0.05)., Conclusion: The magnetic c-erbB-2 antisense probe can effectively transfect and specifically inhibit the expression of SK-Br-3 cell lines at the iron concentration of 25 mg/L.

Once considered genetic "oddities", microRNAs (miRNAs) are now recognized as key epigenetic regulators of numerous biological processes, including some with a causal link to the pathogenesis, maintenance, and treatment of cancer. The crux of small RNA-based therapeutics lies in the antagonism of potent cellular targets; the main shortcoming of the field in general, lies in ineffective delivery. Inhibition of oncogenic miRNAs is a relatively nascent therapeutic concept, but as with predecessor RNA-based therapies, success hinges on delivery efficacy. This review will describes the canonical (e.g. pharmacokinetics and clearance, cellular uptake, endosome escape, etc.) and non-canonical (e.g. spatial localization and accessibility of miRNA, technical limitations of miRNA inhibition, off-target impacts, etc.) challenges to the delivery of antisense-based anti-miRNA therapeutics (i.e. antimiRs) for the treatment of cancer. Emphasis will be placed on how the current leading antimiR platforms-ranging from naked chemically modified oligonucleotides to nanoscale delivery vehicles-are affected by and overcome these barriers. The perplexity of antimiR delivery presents both engineering and biological hurdles that must be overcome in order to capitalize on the extensive pharmacological benefits of antagonizing tumor-associated miRNAs.

In recent years, the number and types of known functional noncoding RNAs have increased considerably. A subset of both short- and long-sized species are known to be involved in the cis regulation of target genes located at or near the same genomic locus. Their expression is often coordinated with that of neighboring protein-coding genes, and in many cases, related transcripts can influence each other at one step or another during their biogenesis. Here, we review the current literature, summarizing the existing knowledge about mammalian cis-acting RNAs and their impact on physiological and disease states.

Motivation: Genome-wide pervasive transcription is widespread in eukaryotes, revealing an extensive array of antisense transcription that involves hundreds of previously unknown non-coding RNAs. Individual cases have shown that antisense transcription influences sense transcription, however, genome-wide mechanisms of how antisense transcription regulates sense transcription remain to be elucidated., Results: Here, we performed a systematic analysis of sense-antisense transcription and nucleosome occupancy in yeast. We found that antisense transcription is associated with nucleosome occupancy in sense promoters. Using RNA polymerase II inactivation data as a reasonable approximation to antisense transcription inactivation data, we further showed that antisense transcripts increase nucleosome occupancy in sense promoter regions they overlap, and reduce nucleosome occupancy in sense promoter regions around their transcription termination sites. These results reveal the previously unappreciated roles of antisense transcription in directing nucleosome occupancy in sense promoters. Our findings will have implications in understanding regulatory functions of antisense transcription.

We have cloned the apteronotid homologs of FoxP2, Otx1, and FoxO3. There was, in the case of all three genes, good similarity between the apteronotid and human amino acid sequences: FoxP2, 78%; Otx1, 54%; FoxO3, 71%. The functional domains of these genes were conserved to a far greater extent, on average: FoxP2, 89%; Otx1, 76%; FoxO3, 82%. This led us to hypothesize that the cellular functions of these genes might also be conserved. We used in situ hybridization to examine the distribution of the mRNA transcripts of these genes in the apteronotid telencephalon. We confined our analysis to the pallial regions previously associated with learning about social signals, whose circuitry has been closely examined in the other articles of this series. We found that AptFoxP2 and AptOtx1 transcripts were expressed predominantly in the dorsocentral division of the pallium (DC); the dorsolateral division of the pallium (DL) contained only weakly labeled neurons. In both cases, the distribution of labeled neurons was very heterogeneous, and unlabeled neurons could be found adjacent to strongly labeled ones. In contrast, we found that most neurons in DL strongly expressed AptFoxO3 mRNA, although there was only weak expression in a small number of cells within DC. We briefly discuss the relevance of our results regarding the functional roles of AptFoxP2/AptOtx1-expressing neurons in DC for communication vs. foraging behavior. We extensively discuss the implications of our results for possible homologies between DL and DC and medial and dorsal pallium of tetrapods, respectively., (Copyright © 2012 Wiley Periodicals, Inc.)

An antisense construct targeting the C(4) isoform of NADP-malic enzyme (ME), the primary enzyme decarboxylating malate in bundle sheath cells to supply CO(2) to Rubisco, was used to transform the dicot Flaveria bidentis. Transgenic plants (α-NADP-ME) exhibited a 34% to 75% reduction in NADP-ME activity relative to the wild type with no visible growth phenotype. We characterized the effect of reducing NADP-ME on photosynthesis by measuring in vitro photosynthetic enzyme activity, gas exchange, and real-time carbon isotope discrimination (Δ). In α-NADP-ME plants with less than 40% of wild-type NADP-ME activity, CO(2) assimilation rates at high intercellular CO(2) were significantly reduced, whereas the in vitro activities of both phosphoenolpyruvate carboxylase and Rubisco were increased. Δ measured concurrently with gas exchange in these plants showed a lower Δ and thus a lower calculated leakiness of CO(2) (the ratio of CO(2) leak rate from the bundle sheath to the rate of CO(2) supply). Comparative measurements on antisense Rubisco small subunit F. bidentis plants showed the opposite effect of increased Δ and leakiness. We use these measurements to estimate the C(4) cycle rate, bundle sheath leak rate, and bundle sheath CO(2) concentration. The comparison of α-NADP-ME and antisense Rubisco small subunit demonstrates that the coordination of the C(3) and C(4) cycles that exist during environmental perturbations by light and CO(2) can be disrupted through transgenic manipulations. Furthermore, our results suggest that the efficiency of the C(4) pathway could potentially be improved through a reduction in C(4) cycle activity or increased C(3) cycle activity.

Fatty acid synthesis is a coordinated process involving multiple enzymes. Overexpression of some of these enzymes plays important roles in tumor growth and development. Therefore, these enzymes are attractive targets for cancer therapies. Antisense agents provide highly specific inhibition of the expression of target genes and thus have served as powerful tools for gene functional studies and potential therapeutic agents for cancers. This article reviews different types of antisense agents and their applications in the modulation of fatty acid synthesis. Patents of antisense agents targeting fatty acid synthetic enzymes are introduced. In addition, miR-122 has been shown to regulate the expression of fatty acid synthetic enzymes, and thus antisense agent patents that inhibit miR-122 expression are also discussed.

Vivo-morpholinos are commercially available morpholino oligomers with a terminal octa-guanidinium dendrimer for enhanced cell-permeability. Existing evidence from systemically delivered vivo-morpholinos indicate that genetic suppression can last from days to weeks without evidence of cellular toxicity. However, intravenously delivered vivo-morpholinos are ineffective at protein suppression in the brain, and no evidence is available regarding whether intracranially delivered vivo-morpholinos effectively reduce target protein levels, or do so without inducing neurotoxicity. Here we report examples in which in vivo microinjection of antisense vivo-morpholinos directed against three different targets (xCT, GLT1, orexin) in two different brain regions resulted in significant suppression of protein expression without neurotoxicity. Expression was significantly suppressed at six to seven days post-administration, but returned to baseline levels within fourteen days. These results indicate that direct intracranial administration of vivo-morpholinos provides an effective means by which to suppress protein expression in the brain for one to two weeks., (Copyright © 2011 Elsevier B.V. All rights reserved.)

Many inherited diseases are associated with changed splicing patterns, and alternative splicing influences several biological processes as well as the replication of certain viral pathogens. For this reason, there is a broad interest in modulating individual splicing events for therapeutic purposes. Based on the small nuclear RNA (snRNA) U7, we have developed expression vectors for short antisense RNAs that accumulate in the cell nucleus where splicing occurs and that can very specifically modulate the splicing of individual exons. More specifically, in the context of the fatal neuromuscular disorder Spinal Muscular Atrophy (SMA), we have shown that U7 snRNA constructs can restore the inclusion of exon 7 in the SMN2 gene and thereby alleviate or even fully cure disease symptoms in a severe mouse model for SMA. Here we describe more generally procedures to produce U7 constructs to induce exon inclusion and to test their efficiency in cell culture experiments at the level of RNA as well as protein. The analytical methods comprise reverse transcription (RT-)PCR to detect the splicing changes, quantitative real-time RT-PCR to measure U7 snRNA expression levels and western blot and immunofluorescence methods to detect a restoration of protein expression. Additionally, we indicate how U7 cassettes can be introduced into gene transfer vectors for in vivo experiments in animal models or to transduce cell systems that are not readily amenable to DNA transfection.

Despite considerable progress understanding genes that affect the HDL particle, its function, and cholesterol content, genes identified to date explain only a small percentage of the genetic variation. We used N-ethyl-N-nitrosourea mutagenesis in mice to discover novel genes that affect HDL cholesterol levels. Two mutant lines (Hlb218 and Hlb320) with low HDL cholesterol levels were established. Causal mutations in these lines were mapped using linkage analysis: for line Hlb218 within a 12 Mbp region on Chr 10; and for line Hlb320 within a 21 Mbp region on Chr 7. High-throughput sequencing of Hlb218 liver RNA identified a mutation in Pla2g12b. The transition of G to A leads to a cysteine to tyrosine change and most likely causes a loss of a disulfide bridge. Microarray analysis of Hlb320 liver RNA showed a 7-fold downregulation of Hpn; sequencing identified a mutation in the 3' splice site of exon 8. Northern blot confirmed lower mRNA expression level in Hlb320 and did not show a difference in splicing, suggesting that the mutation only affects the splicing rate. In addition to affecting HDL cholesterol, the mutated genes also lead to reduction in serum non-HDL cholesterol and triglyceride levels. Despite low HDL cholesterol levels, the mice from both mutant lines show similar atherosclerotic lesion sizes compared to control mice. These new mutant mouse models are valuable tools to further study the role of these genes, their affect on HDL cholesterol levels, and metabolism.

Here we present a protocol, which allows loss-of-function studies in Xenopus embryos using antisense morpholino oligonucleotides (MOs). Gene knockdown studies provide a critical method for assessing gene function in vitro and in vivo. Such studies are currently performed in Xenopus using primarily one of the two main methods: (1) overexpression of dominant negative constructs or (2) inhibition of gene function by using MOs targeting either the initiation of translation or mRNA splicing. While a dominant negative approach is very effective, it often suffers from specificity. Given that MOs target very specific nucleotide sequences in the target RNA, it suffers considerably less from issues of specificity. The most convenient method for introducing MOs into embryos is through microinjection, which is a simple procedure. Therefore, a reverse genetics approach in Xenopus using MOs is an extremely powerful tool to study gene function, particularly when taking advantage of available sequence data in the post-genomic era. Furthermore, given the well-established fate map in Xenopus, it is also very easy to generate mosaic knockdown embryos, where the gene of interest is affected in defined regions of the embryo. Finally it should be noted that MOs can also be used to block miRNA function and processing, so that it provides a convenient method to not only perform gene knockdown studies on protein coding genes, but also noncoding genes. The protocol we describe here is for both Xenopus laevis and Xenopus tropicalis.

Although behavioural experiments demonstrate that colouration influences mate choice in many species, a complete understanding of this form of signalling requires information about colour vision in the species under investigation. The guppy (Poecilia reticulata) has become a model species for the study of colour-based sexual selection. To investigate the role of opsin gene duplication and divergence in the evolution of colour-based mate choice, we used in situ hybridization to determine where the guppy's nine cone opsins are expressed in the retina. Long wavelength-sensitive (LWS) opsins were more abundant in the dorsal retina than in the ventral retina. One of the middle wavelength-sensitive opsins (RH2-1) exhibited the opposite pattern, while the other middle wavelength-sensitive opsin (RH2-2) and the short wavelength-sensitive opsins (SWS1, SWS2A and SWS2B) were expressed throughout the retina. We also found variation in LWS opsin expression among individuals. These observations suggest that regions of the guppy retina are specialized with respect to wavelength discrimination and/or sensitivity. Intra-retinal variability in opsin expression, which has been observed in several fish species, might be an adaptation to variation in the strength and spectral composition of light entering the eye from above and below. The discovery that opsin expression varies in the guppy retina may motivate new behavioural experiments designed to study its role in mate choice.

Although studies on the immunopathogenesis of anti-neutrophil cytoplasm antibody (ANCA) vasculitis have been directed at understanding the autoantibody, there is growing evidence that points to the importance of ANCA autoantigen genes and their regulation. Transcriptional analysis indicates that ANCA autoantigen genes are active in mature neutrophils of ANCA vasculitis patients compared to healthy controls. The unusual transcriptional state of neutrophils from ANCA vasculitis patients appears to be a consequence of failed or disrupted epigenetic silencing. Defective epigenetic silencing could have global effects, by altering the transcriptional and phenotypic state of neutrophils, or local effects by permitting transcription of autoantigen genes from both strands resulting in anti-sense transcripts. Although the role of anti-sense transcripts is currently unknown, there are two intriguing possibilities. Anti-sense transcripts could function (as described for other genes) in transcriptional silencing of autoantigen genes, which takes place in normal neutrophil progenitors. In the setting of failed epigenetic silencing, the fate of anti-sense transcripts may be pathological and serve as a template for production of complementary autoantigens. The observation that ANCA vasculitis patients have anti-sense transcripts and antibodies to complementary proteins is consistent with a role of anti-sense transcripts in complementary protein production. A better understanding of epigenetic silencing and complementary proteins in ANCA vasculitis may unlock the underlying pathology of this condition., (© 2011 The Authors; Clinical and Experimental Immunology © 2011 British Society for Immunology.)

The methylation patterns of the MET1 gene in organs of Arabidopsis thaliana were studied by Southern blot hybridization of DNA samples hydrolyzed with differentially methylation-sensitive restriction endonucleases. A highly methylated on internal cytosine residue CCGG site was found 1.5 kb upstream of the gene, whereas CCGG sites located in more proximal parts of the 5'-flanking region and the gene itself are essentially unmethylated. This methylation pattern was observed in different organs of plants belonging to two different ecotypes as well as in different transgenic plant lines. The methylation level ofa CCGG site in exon 3 (2.1 kb from the gene's 5'-end) occurred to be variable between different transgenic plant lines and two ecotypes studied. Transcription levels of the MET1 gene vary slightly in organs of wild-type plants without any obvious correlation with its methylation. The transgenic antisense MET1 constructs expressed in plant genome do affect both MET1 methylation and its transcription but again without any obvious correlation. The comparative investigation of transcription levels of different genes of cytosine DNA methyltransferase family MET (MET1, MET2a, MET2b, MET3) and their methylation patterns shows that there may exist some mechanisms defending the most actively transcribed gene MET1 of this family from methylation mediated silencing. In contrast to DRM2 gene we could not find any adenine methylated GATC sites in the MET1 gene.

Electroporation is potentially a very powerful technique for both in vitro cellular and in vivo drug delivery, particularly relating to oligonucleotides and their analogs for genetic therapy. Using a sensitive and quantitative HeLa cell luciferase RNA interference mRNA splice correction assay with a functional luciferase readout, we demonstrate that parameters such as peptide nucleic acid (PNA) charge and the method of electroporation have dramatic influence on the efficiency of productive delivery. In a suspended cell electroporation system (cuvettes), a positively charged PNA (+8) was most efficiently transferred, whereas charge neutral PNA was more effective in a microtiter plate electrotransfer system for monolayer cells. Surprisingly, a negatively charged (-23) PNA did not show appreciable activity in either system. Findings from the functional assay were corroborated by pulse parameter variations, polymerase chain reaction, and confocal microscopy. In conclusion, we have found that the charge of PNA and electroporation system combination greatly influences the transfer efficiency, thereby illustrating the complexity of the electroporation mechanism.

Background: Identifying associations between genotypes and gene expression levels using microarrays has enabled systematic interrogation of regulatory variation underlying complex phenotypes. This approach has vast potential for functional characterization of disease states, but its prohibitive cost, given hundreds to thousands of individual samples from populations have to be genotyped and expression profiled, has limited its widespread application., Results: Here we demonstrate that genomic regions with allele-specific expression (ASE) detected by sequencing cDNA are highly enriched for cis-acting expression quantitative trait loci (cis-eQTL) identified by profiling of 500 animals in parallel, with up to 90% agreement on the allele that is preferentially expressed. We also observed widespread noncoding and antisense ASE and identified several allele-specific alternative splicing variants., Conclusion: Monitoring ASE by sequencing cDNA from as little as one sample is a practical alternative to expression genetics for mapping cis-acting variation that regulates RNA transcription and processing.

Uncontrolled extracellular matrix production by fibroblasts in response to tissue injury contributes to fibrotic diseases, such as idiopathic pulmonary fibrosis (IPF), a progressive and ultimately fatal process that currently has no cure. Although dysregulation of miRNAs is known to be involved in a variety of pathophysiologic processes, the role of miRNAs in fibrotic lung diseases is unclear. In this study, we found up-regulation of miR-21 in the lungs of mice with bleomycin-induced fibrosis and also in the lungs of patients with IPF. Increased miR-21 expression was primarily localized to myofibroblasts. Administration of miR-21 antisense probes diminished the severity of experimental lung fibrosis in mice, even when treatment was started 5-7 d after initiation of pulmonary injury. TGF-beta1, a central pathological mediator of fibrotic diseases, enhanced miR-21 expression in primary pulmonary fibroblasts. Increasing miR-21 levels promoted, whereas knocking down miR-21 attenuated, the pro-fibrogenic activity of TGF-beta1 in fibroblasts. A potential mechanism for the role of miR-21 in fibrosis is through regulating the expression of an inhibitory Smad, Smad7. These experiments demonstrate an important role for miR-21 in fibrotic lung diseases and also suggest a novel approach using miRNA therapeutics in treating clinically refractory fibrotic diseases, such as IPF.

Background and Objective: The accumulation of eosinophils in airways is an important characteristic of asthma. The process is primarily mediated by interleukin-5 (IL-5) secreted by Th2 lymphocytes. This study explored a new approach to asthma therapy in which allergic rats were transfected with the IL-5 antisense gene delivered by the recombinant adeno-associated virus (rAAV-ASIL-5)., Methods: The viral vector rAAV-ASIL-5 was constructed and the IL-5 antisense gene transfected into allergic rats. The levels of IL-5, IgE, eotaxin and eosinophilic cationic protein (ECP) in sera and bronchoalveolar lavage fluid (BALF) were measured by ELISA. The inflammatory responses in lung tissues were evaluated by histological study., Results: The levels of IL-5 protein in serum and BALF were significantly decreased in the allergic rats treated with rAAV-ASIL-5 (P < 0.05). Serum ovalbumin-specific IgE was reduced in treated rats compared with untreated rats (P < 0.05). rAAV-ASIL-5 treatment also reduced eosinophils in the peripheral blood and BALF, as well as the ECP and eotaxin levels in serum and BALF (P < 0.05). There was significantly less inflammation in the lungs of rAAV-ASIL-5-treated rats than in those of untreated rats. No obvious pathological damage to the kidneys and livers of the rats treated with rAAV was observed., Conclusions: Treatment with rAAV-ASIL-5 inhibited the accumulation of eosinophils and airway inflammation in the rat model of allergic asthma by suppressing IL-5 production. These results suggest that rAAV-ASIL-5-based gene therapy may be used for the treatment of allergic asthma.

Antigen-presenting cells expressing indoleamine 2,3-dioxygenase (IDO) play a critical role in maintaining peripheral tolerance. Strategies to inhibit IDO gene expression and enhance antigen-presenting cell function might improve anti-tumour immunity. Here we have designed highly effective anti-IDO small interfering (si) RNAs that function at low concentrations. When delivered to human primary immune cells such as monocytes and dendritic cells (DCs), they totally inhibited IDO gene expression without impairing DC maturation and function. Depending on the design and chemical modifications, we show that it is possible to design either monofunctional siRNAs devoid of immunostimulation or bifunctional siRNAs with gene silencing and immunostimulatory activities. The latter are able to knockdown IDO expression and induce cytokine production through either endosomal Toll-like receptor 7/8 or cytoplasmic retinoid acid-inducible gene 1 helicase. Inhibition of IDO expression with both classes of siRNAs inhibited DC immunosuppressive function on T-cell proliferation. Immature monocyte-derived DCs that had been transfected with siRNA-bearing 5'-triphosphate activated T cells, indicating that, even in the absence of external stimuli such as tumour necrosis factor-alpha, those DCs were sufficiently mature to initiate T-cell activation. Collectively, our data highlight the potential therapeutic applications of this new generation of siRNAs in immunotherapy.

Background: Recent transcriptomic analyses in mammals have uncovered the widespread occurrence of endogenous antisense transcripts, termed natural antisense transcripts (NATs). NATs are transcribed from the opposite strand of the gene locus and are thought to control sense gene expression, but the mechanism of such regulation is as yet unknown. Although several thousand potential sense-antisense pairs have been identified in mammals, examples of functionally characterized NATs remain limited. To identify NAT candidates suitable for further functional analyses, we performed DNA microarray-based NAT screening using mouse adult normal tissues and mammary tumors to target not only the sense orientation but also the complementary strand of the annotated genes., Results: First, we designed microarray probes to target the complementary strand of genes for which an antisense counterpart had been identified only in human public cDNA sources, but not in the mouse. We observed a prominent expression signal from 66.1% of 635 target genes, and 58 genes of these showed tissue-specific expression. Expression analyses of selected examples (Acaa1b and Aard) confirmed their dynamic transcription in vivo. Although interspecies conservation of NAT expression was previously investigated by the presence of cDNA sources in both species, our results suggest that there are more examples of human-mouse conserved NATs that could not be identified by cDNA sources. We also designed probes to target the complementary strand of well-characterized genes, including oncogenes, and compared the expression of these genes between mammary cancerous tissues and non-pathological tissues. We found that antisense expression of 95 genes of 404 well-annotated genes was markedly altered in tumor tissue compared with that in normal tissue and that 19 of these genes also exhibited changes in sense gene expression. These results highlight the importance of NAT expression in the regulation of cellular events and in pathological conditions., Conclusion: Our microarray platform targeting the complementary strand of annotated genes successfully identified novel NATs that could not be identified by publically available cDNA data, and as such could not be detected by the usual "sense-targeting" microarray approach. Differentially expressed NATs monitored by this platform may provide candidates for investigations of gene function. An advantage of our microarray platform is that it can be applied to any genes and target samples of interest.