A construct with fluorescent indicators for conditional expression of miRNA

<p>Abstract</p> <p>Background</p> <p>Transgenic RNAi holds promise as a simple, low-cost, and fast method for reverse genetics in mammals. It may be particularly useful for producing animal models for hypomorphic gene function. Inducible RNAi that permits spatially and temporally controllable gene silencing in vivo will enhance the power of transgenic RNAi approach. Furthermore, because microRNA (miRNA) targeting specific genes can be expressed simultaneously with protein coding genes, incorporation of fluorescent marker proteins can simplify the screening and analysis of transgenic RNAi animals.</p> <p>Results</p> <p>We sought to optimally express a miRNA simultaneously with a fluorescent marker. We compared two construct designs. One expressed a red fluorescent protein (RFP) and a miRNA placed in its 3' untranslated region (UTR). The other expressed the same RFP and miRNA, but the precursor miRNA (pre-miRNA) coding sequence was placed in an intron that was inserted into the 3'-UTR. We found that the two constructs expressed comparable levels of miRNA. However, the intron-containing construct expressed a significantly higher level of RFP than the intron-less construct. Further experiments indicate that the 3'-UTR intron enhances RFP expression by its intrinsic gene-expression-enhancing activity and by eliminating the inhibitory effect of the pre-miRNA on the expression of RFP. Based on these findings, we incorporated the intron-embedded pre-miRNA design into a conditional expression construct that employed the Cre-loxP system. This construct initially expressed EGFP gene, which was flanked by loxP sites. After exposure to Cre recombinase, the transgene stopped EGFP expression and began expression of RFP and a miRNA, which silenced the expression of specific cellular genes.</p> <p>Conclusion</p> <p>We have designed and tested a conditional miRNA-expression construct and showed that this construct expresses both the marker genes strongly and can silence the target gene efficiently upon Cre-mediated induction of the miRNA expression. This construct can be used to increase the efficiency of making cell lines or transgenic animals that stably express miRNA targeting specific genes.</p

Achieving short-cut nitrification and denitrification in modified intermittently aerated constructed wetland

This study aim to enhance nitrogen removal performance via shifting nitrogen removal pathway from nitrate to nitrite pathway. It was demonstrated that nitrite pathway was successfully and stably achieved in CWs by using modified intermittent aeration control with aeration 20 min/non-aeration 100 min and reducing DO concentration during aeration, nitrite in the effluent could accumulate to over 70% of the total oxidized nitrogen. Q-PCR analysis showed that nitrifying microbial communities were optimized under the alternating anoxic and aerobic conditions, ammonia oxidizing bacteria increased from 7.15 x 10(6) to 8.99 x 10(6) copies/g, while nitrite oxidizing bacteria decreased approximately threefold after 234 days operation. Most importantly, high nitrogen removal efficiency with ammonium removal efficiency of 94.6%, and total nitrogen removal efficiency of 82.6% could be achieved via nitrite pathway even under carbon limiting conditions. In comparison to the nitrate pathway, the nitrite pathway could improve the TN removal by about 55%. (C) 2017 Elsevier Ltd. All rights reserved.</p

The combined effect of dissolved oxygen and COD/N on nitrogen removal and the corresponding mechanisms in intermittent aeration constructed wetlands

In this study, the combined effect of dissolved oxygen (DO) and COD/N on nitrogen (N) removal as well as the corresponding mechanisms were investigated in aerated constructed wetlands (CWs). At each investigated COD/N level, the ammonium removal efficiency increased as DO concentration increased. However, the highest total N removal efficiency occurred at different DO concentration at each COD/N level. The results of functional gene analysis and cyclic N profile studies indicated that DO supply and COD/N influence the N removal performance, which is not only exert a direct effect on nitrification-denitrification process, but also change N removal pathway in intermittent aerated CWs. At a relatively high influent COD/N of 20, the simultaneous nitrification and denitrification (SND) via nitrite was almost the exclusive N removal pathway at all investigated DO concentration. With the decrease of COD/N from 20 to 2 at DO of similar to 1.8, similar to 3.5 and similar to 6.0 mg/L, SND efficiency all decreased, however, its decreasing rate was much higher at relatively high DO level of similar to 6.0 mg/L than that at DO levels of similar to 1.8 and similar to 3.5 mg/L. In comparison, a simultaneously partial nitrification, anammox and denitrification was established at DO of similar to 0.8 mg/L along with reducing influent COD/N

Nerve Injection of Viral Vectors Efficiently Transfers Transgenes into Motor Neurons and Delivers RNAi Therapy Against ALS

RNA interference (RNAi) mediates sequence-specific gene silencing, which can be harnessed to silencing disease-causing genes for therapy. Particularly suitable diseases are those caused by dominant, gain-of-function type of gene mutations. In these diseases, the mutant gene generates a mutant protein or RNA product, which possesses toxic properties that harm cells. By silencing the mutant gene, the toxicity can be lessened because the amount of the toxic product is lowered in cells. In this report, we tested RNAi therapy in a mouse model for amyotrophic lateral sclerosis (ALS), which causes motor neuron degeneration, paralysis, and death. We used a transgenic model that overexpresses mutant Cu, Zn superoxide dismutase (SOD1G93A), which causes ALS by a gained toxic property. We delivered RNAi using recombinant adenovirus (RAd) and adeno-associated virus serotype 2 (AAV2). We compared the efficiency of RNAi delivery between injecting the viral vectors into muscle and into nerve, and found that nerve injetion is more efficient in delivering RNAi to motor neurons. Based on this data, we conducted therapeutic trials in the mouse model and found that nerve injection of RAd, but not AAV2, at the disease onset had a modest therapeutic efficacy. These results highlight the potential and the challenges in delivering RNAi therapy by gene therepy. Antioxid. Redox Signal. 11, 1523–1534

Diet Affects Muscle Quality and Growth Traits of Grass Carp (Ctenopharyngodon idellus): A Comparison Between Grass and Artificial Feed

Fish muscle, the main edible parts with high protein level and low fat level, is consumed worldwide. Diet contributes greatly to fish growth performance and muscle quality. In order to elucidate the correlation between diet and muscle quality, the same batch of juvenile grass carp (Ctenopharyngodon idellus) were divided into two groups and fed with either grass (Lolium perenne, Euphrasia pectinata and Sorghum sudanense) or artificial feed, respectively. However, the different two diets didn't result in significant differences in all the detected water quality parameters (e.g., Tm, pH, DO, NH3/NH4+-N, NO3--N, NO2-, TN, TP, and TOC) between the two experimental groups. After a 4-month culture period, various indexes and expression of myogenic regulatory factor (MRFs) and their related genes were tested. The weight gain of the fish fed with artificial feed (AFG) was nearly 40% higher than the fish fed with grass (GFG). Significantly higher alkaline phosphatase, total cholestrol, high density cholestrol and total protein were detected in GFG as compared to AFG. GFG also showed increased hardness, resilience and shear force in texture profile analysis, with significantly bigger and compact muscle fibers in histologic slices. The fat accumulation was most serious in the abdomen muscle of AFG. Additionally, the expression levels of MyoG, MyoD, IGF-1, and MSTNs were higher, whereas Myf-5, MRF4, and IGF-2 were lower in most positional muscles of GFG as compared to AFG. Overall, these results suggested that feeding grass could promote muscle growth and development by stimulating muscle fiber hypertrophy, as well as significantly enhance the expression of CoL1As. Feeding C. idellus with grass could also improve flesh quality by improving muscle characteristics, enhancing the production of collagen, meanthile, reducing fat accumulation and moisture in muscle, but at the cost of a slower growth

Transplantation of Human Induced Pluripotent Stem Cell-Derived Neural Progenitor Cells Promotes Forelimb Functional Recovery after Cervical Spinal Cord Injury

Locomotor function after spinal cord injury (SCI) is critical for assessing recovery. Currently, available means to improve locomotor function include surgery, physical therapy rehabilitation and exoskeleton. Stem cell therapy with neural progenitor cells (NPCs) transplantation is a promising reparative strategy. Along this line, patient-specific induced pluripotent stem cells (iPSCs) are a remarkable autologous cell source, which offer many advantages including: great potential to generate isografts avoiding immunosuppression; the availability of a variety of somatic cells without ethical controversy related to embryo use; and vast differentiation. In this current work, to realize the therapeutic potential of iPSC-NPCs for the treatment of SCI, we transplanted purified iPSCs-derived NPCs into a cervical contusion SCI rat model. Our results showed that the iPSC-NPCs were able to survive and differentiate into both neurons and astrocytes and, importantly, improve forelimb locomotor function as assessed by the grooming task and horizontal ladder test. Purified iPSC-NPCs represent a promising cell type that could be further tested and developed into a clinically useful cell source for targeted cell therapy for cervical SCI patients

Selective silencing by RNAi of a dominant allele that causes amyotrophic lateral sclerosis

RNA interference (RNAi) can achieve sequence-selective inactivation of gene expression in a wide variety of eukaryotes by introducing double-stranded RNA corresponding to the target gene. Here we explore the potential of RNAi as a therapy for amyotrophic lateral sclerosis (ALS) caused by mutations in the Cu, Zn superoxide dismutase (SOD1) gene. Although the mutant SOD1 is toxic, the wild-type SOD1 performs important functions. Therefore, the ideal therapeutic strategy should be to selectively inhibit the mutant, but not the wild-type SOD1 expression. Because most SOD1 mutations are single nucleotide changes, to selectively silence the mutant requires single-nucleotide specificity. By coupling rational design of small interfering RNAs (siRNAs) with their validation in RNAi reactions in vitro and in vivo, we have identified siRNA sequences with this specificity. A similarly designed sequence, when expressed as small hairpin RNA (shRNA) under the control of an RNA polymerase III (pol III) promoter, retains the single-nucleotide specificity. Thus, RNAi is a promising therapy for ALS and other disorders caused by dominant, gain-of-function gene mutations

Transition metal-catalysed molecular n-doping of organic semiconductors

Electron doping of organic semiconductors is typically inefficient, but here a precursor molecular dopant is used to deliver higher n-doping efficiency in a much shorter doping time. Chemical doping is a key process for investigating charge transport in organic semiconductors and improving certain (opto)electronic devices(1-9). N(electron)-doping is fundamentally more challenging than p(hole)-doping and typically achieves a very low doping efficiency (eta) of less than 10%(1,10). An efficient molecular n-dopant should simultaneously exhibit a high reducing power and air stability for broad applicability(1,5,6,9,11), which is very challenging. Here we show a general concept of catalysed n-doping of organic semiconductors using air-stable precursor-type molecular dopants. Incorporation of a transition metal (for example, Pt, Au, Pd) as vapour-deposited nanoparticles or solution-processable organometallic complexes (for example, Pd-2(dba)(3)) catalyses the reaction, as assessed by experimental and theoretical evidence, enabling greatly increased eta in a much shorter doping time and high electrical conductivities (above 100 S cm(-1); ref. (12)). This methodology has technological implications for realizing improved semiconductor devices and offers a broad exploration space of ternary systems comprising catalysts, molecular dopants and semiconductors, thus opening new opportunities in n-doping research and applications(12, 13)

Dialkoxybithiazole: A New Building Block for Head-to-Head Polymer Semiconductors

Polymer semiconductors have received great attention for organic electronics due to the low fabrication cost offered by solution-based printing techniques. To enable the desired solubility/processability and carrier mobility, polymers are functionalized with hydrocarbon chains by strategically manipulating the alkylation patterns. Note that head-to-head (HH) linkages have traditionally been avoided because the induced backbone torsion leads to poor π–π overlap and amorphous film microstructures, and hence to low carrier mobilities. We report here the synthesis of a new building block for HH linkages, 4,4′-dialkoxy-5,5′-bithiazole (<b>BTzOR</b>), and its incorporation into polymers for high performance organic thin-film transistors. The small oxygen van der Waals radius and intramolecular S­(thiazolyl)···O­(alkoxy) attraction promote HH macromolecular architectures with extensive π-conjugation, low bandgaps (1.40–1.63 eV), and high crystallinity. In comparison to previously reported 3,3′-dialkoxy-2,2′-bithiophene (<b>BTOR</b>), <b>BTzOR</b> is a promising building block in view of thiazole geometric and electronic properties: (a) replacing (thiophene)­C–H with (thiazole)N reduces steric encumbrance in <b>–BTzOR–Ar–</b> dyads by eliminating repulsive C–H···H–C interactions with neighboring arene units, thereby enhancing π–π overlap and film crystallinity; and (b) thiazole electron-deficiency compensates alkoxy electron-donating characteristics, thereby lowering the <b>BTzOR</b> polymer HOMO versus that of the <b>BTOR</b> analogues. Thus, the new <b>BTzOR</b> polymers show substantial hole mobilities (0.06–0.25 cm²/(V s)) in organic thin-film transistors, as well as enhanced <i>I</i>_on:<i>I</i>_off ratios and greater ambient stability than the <b>BTOR</b> analogues. These geometric and electronic properties make <b>BTzOR</b> a promising building block for new classes of polymer semiconductors, and the synthetic route to <b>BTzOR</b> reported here should be adaptable to many other bithiazole-based building blocks