RNA‐based therapeutic approaches for coagulation factor deficiencies

Summary. Substitutive therapy has significantly ameliorated the quality of life of patients with coagulation factor deficiencies. However, there are some limitations that support research towards alternative therapeutic approaches. Here we focus on the rescue of coagulation factor biosynthesis by targeting the RNA processing and translation, which would permit restoration of the altered gene expression while maintaining the gene regulation in the physiological tissues. The essential prerequisite of the three reported RNA‐based correction approaches (i–iii), which rely on mutation types and are applicable even to large size mRNAs, is the presence in cells of the precursor (pre‐mRNA) or mature mRNA forms. (i) In the F7 gene, modification of the small nuclear RNA U1 (U1 snRNA), the key component of the spliceosomal U1 ribonucleoprotein, re‐directs correct usage of a mutated exon–intron junction, triggering synthesis of correct mRNA and secretion of functional factor (F)VII. (ii) Spliceosome‐mediated RNA trans‐splicing (SMaRT) between mutated and engineered pre‐mRNAs produces normal FVIII mRNA and secretion of functional protein. (iii) Aminoglycoside drugs induce ribosome readthrough and suppress premature translation termination caused by nonsense mutations in FVII, VIII and IX. The rescued expression levels ranged from very low (aminoglycosides) to moderate (U1 snRNA and SMaRT), which could result in amelioration of the disease phenotypes. These findings prompt further studies aimed at demonstrating the clinical translatability of RNA‐based strategies, which might open new avenues in the treatment of coagulation factor deficiencies.     

The nuclear hormone receptor E75A regulates vitellogenin gene (Al‐Vg) expression in the mirid bug Apolygus lucorum

Apolygus lucorum is the predominant pest of Bacillus thuringiensis (Bt) cotton in China. 20‐hydroxyecdysone (20E) plays a key role in the reproduction of this insect. To better understand the mechanism underlying 20E‐regulated reproduction, the nuclear hormone receptor E75 isoform‐A of Ap. lucorum (Al‐E75A) was cloned and its expression analysed. A 2241‐bp sequence of Al‐E75A cDNA encoded an open reading frame of a polypeptide with a predicted molecular mass of 69.04 kDa. Al‐E75A mRNA was detected in female adult stages of Ap. lucorum with peak expression in 7‐day‐old animals. Al‐E75A was also expressed in several tissues, particularly in the fat body and ovary. A 3.2 kb Al‐E75A mRNA was detected in all tissues by Northern blot. The fecundity and longevity were significantly decreased in female adults treated with Al‐E75A small interfering RNA. The rates of egg incubation rates were considerably lower in the RNA interference‐treated animals compared to the untreated controls. In order to investigate the molecular mechanism underlying the effects described above, vitellogenin (Al‐Vg) was selected for further investigation. The expression pattern of Al‐Vg was similar to that of Al‐E75A and was up‐regulated by 20E. After knockdown of Al‐E75A, the expression profile of Al‐Vg and the protein levels were down‐regulated. These findings suggest that Al‐E75A plays a crucial role in the regulation of Al‐Vg expression in Ap. lucorum.     

Angiostatin K1‐3 induces E‐selectin via AP1 and Ets1: a mediator for anti‐angiogenic action of K1‐3

Summary. Background: Angiostatin, a circulating angiogenic inhibitor, is an internal fragment of plasminogen and consists of several isoforms, K1‐3 included. We previously showed that K1‐3 was the most potent angiostatin to induce E‐selectin mRNA expression. The purpose of this study was to identify the mechanism responsible for K1‐3‐induced E‐selectin expression and investigate the role of E‐selectin in the anti‐angiogenic action of K1‐3. Methods and results: Quantitative real time RT‐PCR and Western blotting analyses confirmed a time‐dependent increase of E‐selectin mRNA and protein induced by K1‐3. Subcellular fractionation and immunofluorescence microscopy showed the co‐localization of K1‐3‐induced E‐selectin with caveolin 1 (Cav1) in lipid rafts in which E‐selectin may behave as a signaling receptor. Promoter‐driven reporter assays and site‐directed mutagenesis showed that K1‐3 induced E‐selectin expression via promoter activation and AP1 and Ets‐1 binding sites in the proximal E‐selectin promoter were required for E‐selectin induction. The in vivo binding of both protein complexes to the proximal promoter was confirmed by chromatin immunoprecipitation (ChIP). Although K1‐3 induced the activation of ERK1/2 and JNK, only repression of JNK activation attenuated the induction of E‐selectin by K1‐3. A modulatory role of E‐selectin in the anti‐angiogenic action of K1‐3 was manifested by both overexpression and knockdown of E‐selectin followed by cell proliferation assay. Conclusions: We show that K1‐3 induced E‐selectin expression via AP1 and Ets‐1 binding to the proximal E‐selectin promoter (?356/+1), which was positively mediated by JNK activation. Our findings also demonstrate E‐selectin as a novel target for the anti‐angiogenic therapy.     

Tetracycline‐suppressible female lethality and sterility in the Mexican fruit fly,Anastrepha ludens

The sterile insect technique (SIT) involves the mass release of sterile males to suppress insect pest populations. SIT has been improved for larval pests by the development of strains for female‐specific tetracycline‐suppressible (Tet‐off) embryonic lethal systems for male‐only populations. Here we describe the extension of this approach to the Mexican fruit fly, Anastrepha ludens, using a Tet‐off driver construct with the Tet‐transactivator (tTA) under embryo‐specific Anastrepha suspensa serendipity α (As‐sry‐ α ) promoter regulation. In the absence of tetracycline, tTA acts upon a Tet‐response element linked to the pro‐apoptotic cell death gene lethal effector, head involuation defective (hid), from A. ludens (Alhid^Ala2) that contains a sex‐specific intron splicing cassette, resulting in female‐specific expression of the lethal effector. Parental adults double‐homozygous for the driver/effector vectors were expected to yield male‐only progeny when reared on Tet‐free diet, but a complete lack of oviposited eggs resulted for each of the three strains tested. Ovary dissection revealed nonvitellogenic oocytes in all strains that was reversible by feeding females tetracycline for 5 days after eclosion, resulting in male‐only adults in one strain. Presumably the sry‐ α promoter exhibits prezygotic maternal expression as well as zygotic embryonic expression in A. ludens, resulting in a Tet‐off sterility effect in addition to female‐specific lethality.     

20‐Hydroxyecdysone stimulates nuclear accumulation of BmNep1, a nuclear ribosome biogenesis‐related protein in the silkworm,Bombyx mori

The pathway of communication between endocrine hormones and ribosome biogenesis critical for physiological adaptation is largely unknown. Nucleolar essential protein 1 (Nep1) is an essential gene for ribosome biogenesis and is functionally conserved in many in vertebrate and invertebrate species. In this study, we cloned Bombyx mori Nep1 (BmNep1) due to its high expression in silk glands of silkworms on day 3 of the fifth instar. We found that BmNep1 mRNA and protein levels were upregulated in silk glands during fourth‐instar ecdysis and larval–pupal metamorphosis. By immunoprecipitation with the anti‐BmNep1 antibody and liquid chromatography‐tandem mass spectrometry analyses, it was shown that BmNep1 probably interacts with proteins related to ribosome structure formation. Immunohistochemistry, biochemical fractionation and immunocytochemistry revealed that BmNep1 is localized to the nuclei in Bombyx cells. Using BmN cells originally derived from ovaries, we demonstrated that 20‐hydroxyecdysone (20E) induced BmNep1 expression and stimulated nuclear accumulation of BmNep1. Under physiological conditions, BmNep1 was also upregulated in ovaries during larval–pupal metamorphosis. Overall, our results indicate that the endocrine hormone 20E facilitates nuclear accumulation of BmNep1, which is involved in nuclear ribosome biogenesis in Bombyx.     

An experimental burn wound‐healing study of non‐thermal atmospheric pressure microplasma jet arrays

In contrast with a thermal plasma surgical instrument based on coagulative and ablative properties, low‐temperature (non‐thermal) non‐equilibrium plasmas are known for novel medicinal effects on exposed tissue while minimizing undesirable tissue damage. In this study we demonstrated that arrays of non‐thermal microplasma jet devices fabricated from a transparent polymer can efficiently inactivate fungi (Candida albicans) as well as bacteria (Escherichia coli), both in vitro and in vivo, and that this leads to a significant wound‐healing effect. Microplasma jet arrays offer several advantages over conventional single‐jet devices, including superior packing density, inherent scalability for larger treatment areas, unprecedented material flexibility in a plasma jet device, and the selective generation of medically relevant reactive species at higher plasma densities. The therapeutic effects of our multi‐jet device were verified on second‐degree burns in animal rat models. Reduction of the wound area and the histology of the wound after treatment have been investigated, and expression of interleukin (IL)‐1α, ‐6 and ‐10 was verified to evaluate the healing effects. The consistent effectiveness of non‐thermal plasma treatment has been observed especially in decreasing wound size and promoting re‐epithelialization through collagen arrangement and the regulation of expression of inflammatory genes. Copyright © 2015 John Wiley & Sons, Ltd.     

Maternal provision of non‐sex‐specific transformer messenger RNA in sex determination of the wasp Asobara tabida

In many insect species maternal provision of sex‐specifically spliced messenger RNA (mRNA) of sex determination genes is an essential component of the sex determination mechanism. In haplodiploid Hymenoptera, maternal provision in combination with genomic imprinting has been shown for the parasitoid Nasonia vitripennis, known as maternal effect genomic imprinting sex determination (MEGISD). Here, we characterize the sex determination cascade of Asobara tabida, another hymenopteran parasitoid. We show the presence of the conserved sex determination genes doublesex (dsx), transformer (tra) and transformer‐2 (tra2) orthologues in As. tabida. Of these, At‐dsx and At‐tra are sex‐specifically spliced, indicating a conserved function in sex determination. At‐tra and At‐tra2 mRNA is maternally provided to embryos but, in contrast to most studied insects, As. tabida females transmit a non‐sex‐specific splice form of At‐tra mRNA to the eggs. In this respect, As. tabida sex determination differs from the MEGISD mechanism. How the paternal genome can induce female development in the absence of maternal provision of sex‐specifically spliced mRNA remains an open question. Our study reports a hitherto unknown variant of maternal effect sex determination and accentuates the diversity of insect sex determination mechanisms.