Rig1 receptor plays a critical role in cardiac reprogramming via YY1 signaling.

We discovered that innate immunity plays an important role in the reprogramming of fibroblasts into cardiomyocytes. In this report, we define the role of a novel retinoic acid-inducible gene 1 Yin Yang 1 (Rig1:YY1) pathway. We found that fibroblast to cardiomyocyte reprogramming efficacy was enhanced by specific Rig1 activators. To understand the mechanism of action, we performed various transcriptomic, nucleosome occupancy, and epigenomic approaches. Analysis of the datasets indicated that Rig1 agonists had no effect on reprogramming-induced changes in nucleosome occupancy or loss of inhibitory epigenetic motifs. Instead, Rig1 agonists were found to modulate cardiac reprogramming by promoting the binding of YY1 specifically to cardiac genes. To conclude, these results show that the Rig1:YY1 pathway plays a critical role in fibroblast to cardiomyocyte reprogramming.

Enhancing cardiac reprogramming via synthetic RNA oligonucleotides.

Reprogramming scar fibroblasts into new heart muscle cells has the potential to restore function to the injured heart. However, the effectiveness of reprogramming is notably low. We have recently demonstrated that the effectiveness of reprogramming fibroblasts into heart muscle cells (cardiomyocytes) is increased by the addition of RNA-sensing receptor ligands. Clinical use of these ligands is problematic due to their ability to induce adverse inflammatory events. To overcome this issue, we sought to determine whether synthetic analogs of natural RNA-sensing receptor ligands, which avoid generating inflammatory insults and are nuclease resistant, would similarly enhance fibroblast reprogramming into cardiomyocytes. Indeed, one such stabilized RNA, ICR2, increased the expression of cardiomyocyte-specific mRNAs in reprogrammed fibroblasts. Moreover, ICR2 enhanced the ability of reprogramming factors to produce cardiomyocytes with mature sarcomeres. Knockdown assays indicated that the effects of ICR2 were mediated by the RNA-sensing receptors Rig-I and TLR3. In addition, ICR2 reduced the effective dose and number of reprogramming factors needed for efficient reprogramming. In summary, the synthetic RNA oligonucleotide ICR2 is a potential therapeutic agent to enhance cardiac reprogramming efficiency.

Optimizing delivery for efficient cardiac reprogramming.

Following heart injury, cardiomyocytes, are lost and are not regenerated. In their place, fibroblasts invade the dead tissue where they generate a scar, which reduces cardiac function. We and others have demonstrated that combinations of specific miRNAs (miR combo) or transcription factors (GMT), delivered by individual lenti-/retro-viruses in vivo, can convert fibroblasts into cardiomyocytes and improve cardiac function. However, the effects are relatively modest due to the low efficiency of delivery of miR combo or GMT. We hypothesized that efficiency would be improved by optimizing delivery. In the first instance, we developed a multicistronic system to express all four miRNAs of miR combo from a single construct. The order of each miRNA in the multicistronic construct gave rise to different levels of miRNA expression. A combination that resulted in equivalent expression levels of each of the four miRNAs of miR combo showed the highest reprogramming efficiency. Further efficiency can be achieved by directly targeting fibroblasts. Screening of several AAV serotypes indicated that AAV1 displayed tropism towards cardiac fibroblasts. Combining multicistronic expression with AAV1 delivery robustly reprogrammed cardiac fibroblasts into cardiomyocytes in vivo.

Lamins Organize the Global Three-Dimensional Genome from the Nuclear Periphery.

Lamins are structural components of the nuclear lamina (NL) that regulate genome organization and gene expression, but the mechanism remains unclear. Using Hi-C, we show that lamins maintain proper interactions among the topologically associated chromatin domains (TADs) but not their overall architecture. Combining Hi-C with fluorescence in situ hybridization (FISH) and analyses of lamina-associated domains (LADs), we reveal that lamin loss causes expansion or detachment of specific LADs in mouse ESCs. The detached LADs disrupt 3D interactions of both LADs and interior chromatin. 4C and epigenome analyses further demonstrate that lamins maintain the active and repressive chromatin domains among different TADs. By combining these studies with transcriptome analyses, we found a significant correlation between transcription changes and the interaction changes of active and inactive chromatin domains These findings provide a foundation to further study how the nuclear periphery impacts genome organization and transcription in development and NL-associated diseases.

MicroRNA-221 sponge therapy attenuates neointimal hyperplasia and improves blood flows in vein grafts.

BACKGROUND: Vein graft failure due to neointimal hyperplasia remains an important and unresolved problem of cardiovascular surgery. MicroRNA-221 (miR-221) has been shown to play a major role in regulating vascular smooth muscle cell (VSMC) proliferation and phenotype transformation. Thus, the purpose of this study is to determine whether adenovirus mediated miR-221 sponge gene therapy could inhibit vein graft neointimal hyperplasia. METHODS: Adenovirus encoding miR-221 sponge (Ad-miR-221-SP) was used to inhibit VSMC proliferation in vitro and neointimal formation in vivo. Expression of miRNA-221 was evaluated in cultured VSMC and in rat vein graft models following transduction with Ad-miR-221-SP, Ad-Control-SP (without miR-221 antisense binding sites), or Ad-GFP (control). To accelerate the transfer of miR-221 sponge gene to the vein grafts, 20% poloxamer F-127 gel was used to extend virus contact time and 0.25% trypsin to increase virus penetration. RESULTS: miR-221 sponges can significantly decrease the expression of miR-221 and proliferation in cultured VSMC. Cellular proliferation rates were significantly reduced in miR-221 sponge treated grafts as compared with controls at 6 weeks after bypass surgery (19.8% versus 43.6%, P=0.0028). miR-221 sponge gene transfer reduced the neointimal area (210.75 ± 24.13 versus 67.01 ± 12.02, P<0.0001), neointimal thickness (171.86 ± 27.87 versus 64.13 ± 16.23, P<0.0001) and neointima/media ratio (0.74 ± 0.21 versus 1.95 ± 0.25, P<0.0001) in vein grafts versus controls. miR-21 sponge treatment was also improved hemodynamics in vein grafts. We have further identified that p27 (Kip1) is a potential target gene of miR-221 in vein grafts. CONCLUSION: miR-221 sponge therapy can significantly reduce miR-221 activity and inhibit neointimal hyperplasia in vein grafts. Locally adventitial delivery of adenoviruses mediated miRNA sponges may be promising gene therapies to prevent vein graft failure.

Direct activation of human and mouse Oct4 genes using engineered TALE and Cas9 transcription factors.

The newly developed transcription activator-like effector protein (TALE) and clustered regularly interspaced short palindromic repeats/Cas9 transcription factors (TF) offered a powerful and precise approach for modulating gene expression. In this article, we systematically investigated the potential of these new tools in activating the stringently silenced pluripotency gene Oct4 (Pou5f1) in mouse and human somatic cells. First, with a number of TALEs and sgRNAs targeting various regions in the mouse and human Oct4 promoters, we found that the most efficient TALE-VP64s bound around -120 to -80 bp, while highly effective sgRNAs targeted from -147 to -89-bp upstream of the transcription start sites to induce high activity of luciferase reporters. In addition, we observed significant transcriptional synergy when multiple TFs were applied simultaneously. Although individual TFs exhibited marginal activity to up-regulate endogenous gene expression, optimized combinations of TALE-VP64s could enhance endogenous Oct4 transcription up to 30-fold in mouse NIH3T3 cells and 20-fold in human HEK293T cells. More importantly, the enhancement of OCT4 transcription ultimately generated OCT4 proteins. Furthermore, examination of different epigenetic modifiers showed that histone acetyltransferase p300 could enhance both TALE-VP64 and sgRNA/dCas9-VP64 induced transcription of endogenous OCT4. Taken together, our study suggested that engineered TALE-TF and dCas9-TF are useful tools for modulating gene expression in mammalian cells.

LIF-dependent primitive neural stem cells derived from mouse ES cells represent a reversible stage of neural commitment.

Primitive neural stem cells (NSCs) define an early stage of neural induction, thus provide a model to understand the mechanism that controls initial neural commitment. In this study, we investigated primitive NSCs derived from mouse embryonic stem cells (ESCs). By genome-wide transcriptional profiling, we revealed their unique signature and depicted the molecular changes underlying critical cell fate transitions during early neural induction at a global level. Together with qRT-PCR analysis, our data illustrated that primitive NSCs retained expression of key pluripotency genes Oct4 and Nanog, while exhibiting repression of other pluripotency-related genes Zscan4, Foxp1 and Dusp9 and up-regulation of neural markers Sox1 and Hes1. The early differentiation feature in primitive NSCs was also supported by their intermediate characters on cell cycle profiles. Moreover, re-plating primitive NSCs back to ESC culture condition could reverse them back to ESC stage, as shown by reversible regulation of marker genes, cell cycle profile changes and enhanced embryoid body formation. In addition, our microarray analysis also identified genes differentially expressed in primitive NSCs, and loss-of-function analysis demonstrated that Hes1 and Ccdc141 play important function at this stage, opening up an opportunity to further understand the regulation of early neural commitment.

A transgenic Bm cell line of piggyBac transposon-derived targeting expression of humanized glycoproteins through N-glycosylation.

Glycoproteins have been implicated in a wide variety of important biochemical and biological functions, including protein stability, immune function, enzymatic function, cellular adhesion and others. Unfortunately, there is no therapeutic protein produced in insect system to date, due to the expressed glycoproteins are paucimannosidic N-glycans, rather than the complex, terminally sialylated N-glycans in mammalian cells. In this paper, we cloned the necessary genes in glycosylation of mammalian cells, such as N-acetylglucosaminyltransferase II (Gn-TII), galactosyltransferases (Gal-Ts), 2,6-Sial-T (ST6 GalII)and 2,3-Sial-T (ST3GalIII), and transformed them to silkworm genome of BmN cell line through transgenesis to establish a transgenic Bm cell line of piggyBac transposon-derived targeting expression of humanized glycoproteins. The study supplied a new insect cell line which is practically to produce "bisected" complex N-glycans like in mammalian cells.

Heterologous expression characteristics of Trichoderma viride endoglucanase V in the silkworm, Bombyx mori L.

Efficient degradation of cellulose needs a synergistic reaction of the cellulolytic enzymes, which include exoglucanases, endoglucanases, and ß-1,4-glucosidase. In this study, we used an improved Bac-to-Bac/BmNPV baculovirus expression system, which lacks the virus-encoded chitinase cathepsin (v-cath) genes of Bombyx mori nucleopolyhedrovirus (BmNPV), to express the endoglucanase V (EG V) gene from Trichoderma viride in silkworm BmN cells and silkworm larvae, and analyzed the characteristics of the recombinant enzyme in silkworm larvae. The result showed that an around 36-kDa protein was visualized in BmN cells at 48 h after the second-generation recombinant mBacmid/BmNPV/EG V baculovirus infection. The crude enzyme extract from the recombinant baculoviruses-infected silkworms exhibited a significant maximum activity at the environmental condition of pH 5.0 and a temperature of 50 °C, and increased 39.86% and 37.76% compared with that from blank mBacmid/BmNPV baculovirus-infected silkworms and normal silkworms, respectively. It was stable at pH range from 5.0 to 10.0 and at temperature range from 40 to 60 °C. The availability of large quantities of EG V that the silkworm provides might greatly facilitate the future research and the potential application in industries.

Cloning and characterization of the Bombyx mori ecdysone oxidase.

The physiological titer of molting hormones in insects depends on relative activities of synthesis and degradation pathways. Ecdysone oxidase (EO) is a key enzyme in the inactivation of ecdysteroid. However, there are only a few reports on ecdysteroid inactivation and its enzymes in silkworm. In this study, we cloned and characterized the Bombyx mori EO (BmEO). The BmEO cDNA contains an ORF of 1,695 bp and the deduced protein sequence contains 564 amino acid residues. The deduced protein sequence contains two functional domains of glucose-methanol-choline oxidoreductase in N-terminal and C-terminal. Comparing the expression levels of BmEO in different tissues, high transcription was mainly present in hemocytes. Reduced expression of this enzyme is expected to lead to pathological accumulation of ecdysone in the hemolymph of silkworm larvae or pupae. Our data show that RNA inference of BmEO transcripts resulted in the accumulation of ecdysteroid and death of larvae or pupae. We infer that EO is a crucial element in the physiology of insect development.

Cloning and expression pattern of 3-dehydroecdysone 3ß-reductase (3DE 3ß-reductase) from the silkworm, Bombyx mori L.

Molting in insects is regulated by molting hormones (ecdysteroids), which are also crucial to insect growth, development, reproduction, etc. Ecdysone was inactivated to 3-dehydroecdysone (3DE) under ecdysone oxidase (EO), and followed by NAD(P)H-dependent irreversible reduction to 3-epiecdysteroid under 3DE 3a-reductase. On the other hand, 3-dehydroecdysone undergoes reversible reduction to ecdysone by 3DE 3ß-reductase in the hemolymph. In this article, we cloned and characterized 3-dehydroecdysone 3ß-reductase (3DE 3ß-reductase) in the different tissues and the developing stage from the silkworm, Bombyx mori L. The B. mori 3DE 3ß-reductase cDNA contains an ORF 972 bp and the deduced protein sequence containing 323 amino acid residues. Analysis showed that the deduced 3DE 3ß-reductase belongs to the aldo-keto reductase (AKR) superfamily, which has the NAD(P)-binding domain, indicating that the function of 3DE 3ß-reductase depends on the existence of NAD(P)H. Using Escherichia coli, a high level expression of a fusion polypeptide band of approx. 40 kDa was observed. The high transcription of 3DE 3ß-reductase was mainly observed in the genitalia and fatty bodies in the third day of the fifth-instar larvae, followed next in the head, epidermis, and hemocytes. The expression of 3DE 3ß-reductase in the early of every instar was lower than that in the late of instar. When the titer of 3DE is low, higher expression of 3DE 3ß-reductase is necessary to maintain the ecdysone titer in body through converting 3DE to ecdysone, while the 3DE titer is high, the expression of 3DE 3ß-reductase showed feedback inhibition.

Expression of Trichoderma viride endoglucanase III in the larvae of silkworm, Bombyx mori L. and characteristic analysis of the recombinant protein.

Endoglucanase is a part of cellulase which hydrolyzes cellulose into glucose. In this study, we cloned endoglucanase III (EG III) gene from Trichoderma viride strain AS 3.3711 using a PCR-based exon splicing method, and expressed EG III recombinant protein in both silkworm BmN cell line and silkworm larvae with an improved Bac-to-Bac/BmNPV mutant baculovirus expression system, which lacks the chiA and v-cath genes of Bombyx mori nucleopolyhedrovirus (BmNPV). The result showed that around 45 kDa protein was visualized in BmN cells at 48 h after the second generation recombinant mBacmid/BmNPV/EG III baculovirus infection. The enzymes from recombinant baculoviruses infected silkworms exhibited significant maximum enzyme activity at the environmental condition of pH 8.0 and temperature 50°C, and increased 20.94 and 19.13% compared with that from blank mBacmid/BmNPV baculoviruses infected silkworms and normal silkworms, respectively. It was stable at pH range from 5.0 to 9.0 and at temperature range from 40 to 60°C. It provided a possibility to generate transgenic silkworms expressing bio-active cellulase, which can catabolize dietary fibers more efficiently, and it might be of great significance for sericulture industry.

Identification and characterization of two chitin-binding proteins from the peritrophic membrane of the silkworm, Bombyx mori L.

The peritrophic membrane (PM) is a semi-permeable lining of the insect midgut, broadly analogous to the mucous lining of vertebrate gut. The PM proteins are important achievements for the function of the PM. In this study, two chitin-binding proteins (BmPM-P43 and BmPM-P41) from the PM of the silkworm, Bombyx mori, were identified and cloned. These proteins showed the molecular mass of 43 and 41 kDa, respectively. The deduced amino acid sequences codes for a protein of 381 amino acid residues and 364 amino acid residues, containing 12 and 14 cysteine residues followed by similar domain, both of them have 5 cysteine residues in similar position in the C-terminal. The confirmation of these proteins was performed by western blot analysis of recombinant BmPM-P43 and BmPM-P41. The chitin-binding activity analysis showed that the BmPM-P43 and BmPM-P41 could bind to chitin strongly. It is concluded that BmPM-P43 and BmPM-P41 contains a polysaccharide deacetylase domain instead of peritrophin domain, indicated that these two proteins may belong to a new chitin-binding protein family.

Cloning and expression of a cellulase gene in the silkworm, Bombyx mori by improved Bac-to-Bac/BmNPV baculovirus expression system.

Cellulases catalyze the hydrolysis of cellulose which are mainly three types: endoglucanases, cellobiohydrolases and ß-glucosidases. It can be used in converting cellulosic biomass to glucose that can be used in different applications such as production of fuel ethanol, animal feed, waste water treatment and in brewing industry. In this paper, we cloned a 1380-bp endoglucanase I (EG I) gene from mycelium of filamentous fungus Trichoderma viride strain AS 3.3711 using PCR-based exon splicing methods, and expressed the recombinant EG I mature peptide protein in both silkworm BmN cell line and silkworm larvae with a newly established Bac-to-Bac/BmNPV mutant baculovirus expression system, which lacks the virus-encoded chitinase (chiA) and cathepsin (v-cath) genes of Bombyx mori nucleopolyhedrovirus (BmNPV). An around 49-kDa protein was visualized after mBacmid/BmNPV/EG I infection, and the maximum expression in silkworm larvae was at 84 h post-infection. The ANOVA showed that the enzymes from recombinant baculoviruses infected silkworms exhibited significant maximum enzyme activity at the environmental condition of pH 7.0 and temperature 50°C. It was stable at pH range from 5.0 to 10.0 and at temperature range from 50 to 60°C, and increased 24.71 and 22.84% compared with that from wild baculoviruses infected silkworms and normal silkworms, respectively. The availability of large quantities of EG I that the silkworm provides maybe greatly facilitate the future research and the potential application in industries.