Translational Block Research Articles

IL-1β Induces Human Endothelial Surface Expression of IL-15 by Relieving let-7c-3p Suppression of Protein Translation.

Expression of IL-15 on the surface of human graft endothelial cells (ECs) bound to the IL-15Rα subunit can increase the activation of CTLs, potentiating allograft rejection. Our previous work showed that surface expression of this protein complex could be induced by alloantibody-mediated complement activation through increased IL-1β synthesis, secretion, and autocrine/paracrine IL-1-mediated activation of NF-κB. In this article, we report that cultured human ECs express eight differently spliced IL-15 transcripts. Remarkably, IL-1β does not alter the expression level of any IL-15 transcript but induces surface expression independently of RNA polymerase II-mediated transcription while requiring new protein translation. Mechanistically, IL-1β causes an NF-κB-mediated reduction in the level of microRNA Let-7c-3p, thereby relieving a block of translation of IL-15 surface protein. Let7c-3p anti-miR can induce EC surface expression of IL-15/IL-15Rα in the absence of complement activation or of IL-1, enabling IL-15 transpresentation to boost CD8 T cell activation. Because of the complexity we have uncovered in IL-15 regulation, we recommend caution in interpreting increased total IL-15 mRNA or protein levels as a surrogate for transpresentation.

Development of bioconjugate-based delivery systems for nucleic acids.

Nucleic acids are a class of drugs that can modulate gene and protein expression by various mechanisms, namely, RNAi, mRNA degradation by RNase H cleavage, splice modulation, and steric blocking of protein binding or mRNA translation, thus exhibiting immense potential to treat various genetic and rare diseases. Unlike protein-targeted therapeutics, the clinical use of nucleic acids relies on Watson-Crick sequence recognition to regulate aberrant gene expression and impede protein translation. Though promising, targeted delivery remains a bottleneck for the clinical adoption of nucleic acid-based therapeutics. To overcome the delivery challenges associated with nucleic acids, various chemical modifications and bioconjugation-based delivery strategies have been explored. Currently, liver targeting by N-acetyl galactosamine (GalNAc) conjugation has been at the forefront for the treatment of rare and various metabolic diseases, which has led to FDA approval of four nucleic acid drugs. In addition, various other bioconjugation strategies have been explored to facilitate active organ and cell-enriched targeting. This review briefly covers the different classes of nucleic acids, their mechanisms of action, and their challenges. We also elaborate on recent advances in bioconjugation strategies in developing a diverse set of ligands for targeted delivery of nucleic acid drugs.

NRF2 translation block by inhibition of cap-dependent initiation sensitizes lymphoma cells to ferroptosis and CAR-T immunotherapy.

Cancers coopt stress-response pathways to drive oncogenesis, dodge immune surveillance, and resist cytotoxic therapies. Several of these provide protection from ferroptosis, iron-mediated oxidative cell death. Here, we found dramatic sensitization to ferroptosis upon disruption of cap-dependent translation in diffuse large B-cell lymphoma (DLBCL). Specifically, rocaglate inhibitors of the eIF4A1 RNA helicase synergized with pharmacologic ferroptosis inducers, driven by a collapse of glutathione production that protects polyunsaturated fatty acids from ferroptotic oxidation. These effects occur despite initial up-regulation of specific protective factors. We find lost translation of NRF2, oncogenic master regulator of antioxidant gene-expression, is a key consequence of eIF4A1 inhibition. In vivo, combination of the clinical rocaglate zotatifin with a pharmacologically optimized ferroptosis inducer eradicated DLBCL patient derived xenografts. Moreover, we found zotatifin pre-exposure sensitized DLBCL to CD19-directed chimeric antigen receptor (CAR-19) T cells. Translational disruption therefore provides new opportunities to leverage therapeutic impacts of ferroptosis inducers including cytotoxic immunotherapies.

Inhibition of the eukaryotic initiation factor-2α kinase PERK decreases risk of autoimmune diabetes in mice.

Preventing the onset of autoimmune type 1 diabetes (T1D) is feasible through pharmacological interventions that target molecular stress-responsive mechanisms. Cellular stresses, such as nutrient deficiency, viral infection, or unfolded proteins, trigger the integrated stress response (ISR), which curtails protein synthesis by phosphorylating eukaryotic translation initiation factor-2α (eIF2α). In T1D, maladaptive unfolded protein response (UPR) in insulin-producing β cells renders these cells susceptible to autoimmunity. We found that inhibition of the eIF2α kinase PKR-like ER kinase (PERK), a common component of the UPR and ISR, reversed the mRNA translation block in stressed human islets and delayed the onset of diabetes, reduced islet inflammation, and preserved β cell mass in T1D-susceptible mice. Single-cell RNA-Seq of islets from PERK-inhibited mice showed reductions in the UPR and PERK signaling pathways and alterations in antigen-processing and presentation pathways in β cells. Spatial proteomics of islets from these mice showed an increase in the immune checkpoint protein programmed death-ligand 1 (PD-L1) in β cells. Golgi membrane protein 1, whose levels increased following PERK inhibition in human islets and EndoC-βH1 human β cells, interacted with and stabilized PD-L1. Collectively, our studies show that PERK activity enhances β cell immunogenicity and that inhibition of PERK may offer a strategy for preventing or delaying the development of T1D.

Inhibition of the Eukaryotic Initiation Factor-2-α Kinase PERK Decreases Risk of Autoimmune Diabetes in Mice.

Preventing the onset of autoimmune type 1 diabetes (T1D) is feasible through pharmacological interventions that target molecular stress-responsive mechanisms. Cellular stresses, such as nutrient deficiency, viral infection, or unfolded proteins, trigger the integrated stress response (ISR), which curtails protein synthesis by phosphorylating eIF2α. In T1D, maladaptive unfolded protein response (UPR) in insulin-producing β cells renders these cells susceptible to autoimmunity. We show that inhibition of the eIF2α kinase PERK, a common component of the UPR and ISR, reverses the mRNA translation block in stressed human islets and delays the onset of diabetes, reduces islet inflammation, and preserves β cell mass in T1D-susceptible mice. Single-cell RNA sequencing of islets from PERK-inhibited mice shows reductions in the UPR and PERK signaling pathways and alterations in antigen processing and presentation pathways in β cells. Spatial proteomics of islets from these mice shows an increase in the immune checkpoint protein PD-L1 in β cells. Golgi membrane protein 1, whose levels increase following PERK inhibition in human islets and EndoC-βH1 human β cells, interacts with and stabilizes PD-L1. Collectively, our studies show that PERK activity enhances β cell immunogenicity, and inhibition of PERK may offer a strategy to prevent or delay the development of T1D.

Retinoic acid signalling inhibits myogenesis by blocking MYOD translation in pig skeletal muscle cells

Vitamin A is an essential nutrient in animals, playing important roles in animal health. In the pig industry, proper supplementation of vitamin A in the feed can improve pork production performance, while deficiency or excessive intake can lead to growth retardation or disease. However, the specific molecular mechanisms through which vitamin A operates on pig skeletal muscle growth as well as muscle stem cell function remain unexplored. Therefore, in this study, we isolated the pig primary skeletal muscle stem cells (pMuSCs) and treated with retinoic acid (RA), the natural metabolite of vitamin A, and then examined the myogenic capacity of pMuSCs via immunostaining, real-time PCR, CCK8 and western-blot analysis. Unexpectedly, the RA caused a significant decrease in the proliferation and differentiation of pMuSCs. Mechanistically, the RA addition induced the activation of retinoic acid receptor gamma (RARγ), which inhibited the myogenesis through the blockage of protein translation of the master myogenic regulator myogenic differentiation 1 gene (MYOD). Specifically, RARγ inactivate AKT kinase (AKT) signalling and lead to dephosphorylation of eukaryotic translation initiation factor 4E binding protein 1 (eIF4EBP1), which in turn repress the eukaryotic translation initiation factor 4E (eIF4E) complex and block mRNA translation of MYOD. Inhibition of AKT could rescue the myogenic defects of RA-treated pMuSCs. Our findings revealed that retinoid acid signalling inhibits the skeletal muscle stem cell proliferation and differentiation in pigs. Therefore, the vitamin A supplement in the feedstuff should be cautiously optimized to avoid the potential adverse consequences on muscle development associated with the excessive levels of retinoic acid.

An Instruction Inflation Analyzing Framework for Dynamic Binary Translators

Dynamic binary translators (DBTs) are widely used to migrate applications between different instruction set architectures (ISAs). Despite extensive research to improve DBT performance, noticeable overhead remains, preventing near-native performance, especially when translating from complex instruction set computer (CISC) to reduced instruction set computer (RISC). For computational workloads, the main overhead stems from translated code quality. Experimental data show that state-of-the-art DBT products have dynamic code inflation of at least 1.46. This indicates that on average, more than 1.46 host instructions are needed to emulate one guest instruction. Worse, inflation closely correlates with translated code quality. However, the detailed sources of instruction inflation remain unclear. To understand the sources of inflation, we present Deflater , an instruction inflation analysis framework comprising a mathematical model, a collection of black-box unit tests called BenchMIAOes , and a trace-based simulator called InflatSim . The mathematical model calculates overall inflation based on the inflation of individual instructions and translation block optimizations. BenchMIAOes extract model parameters from DBTs without accessing DBT source code. InflatSim implements the model and uses the extracted parameters from BenchMIAOes to simulate a given DBT’s behavior. Deflater is a valuable tool to guide DBT analysis and improvement. Using Deflater, we simulated inflation for three state-of-the-art CISC-to-RISC DBTs: ExaGear, Rosetta2, and LATX, with inflation errors of 5.63%, 5.15%, and 3.44%, respectively for SPEC CPU 2017, gaining insights into these commercial DBTs. Deflater also efficiently models inflation for the open source DBT QEMU and suggests optimizations that can substantially reduce inflation. Implementing the suggested optimizations confirms Deflater’s effective guidance, with 4.65% inflation error, and gains 5.47x performance improvement.

On general extenders in literary translation and all that stuff

Abstract The present article focuses on strategies for translating general extenders (GEs) from English into Romanian. Starting from the generally accepted definition of GEs as structures that extend utterances that are otherwise grammatically complete and that are placed in phrase- or clause-final position, I analyze samples of literary text and their respective (multiple) versions and investigate patterns in which these structures are translated. Since, as pointed out in the literature, GEs can fulfill more than one function in the text, and since in literary texts they tend to be repeatedly and meaningfully employed, the article investigates to what extent a Romanian translator can render this type of pragmatic marker into the target language in a fluid manner. This question is intriguing for at least two reasons: (a) Romanian seems to employ GEs in a more restricted manner than English and (b) repetition seems to be a stumbling block in translation. In order to solve this problem, a translator resorts to lexical variety, compensation, and omission.

Picornavirus 3C Proteins Intervene in Host Cell Processes through Proteolysis and Interactions with RNA.

The Picornaviridae family comprises a large group of non-enveloped viruses with enormous impact on human and animal health. The picornaviral genome contains one open reading frame encoding a single polyprotein that can be processed by viral proteases. The picornaviral 3C proteases share similar three-dimensional structures and play a significant role in the viral life cycle and virus-host interactions. Picornaviral 3C proteins also have conserved RNA-binding activities that contribute to the assembly of the viral RNA replication complex. The 3C protease is important for regulating the host cell response through the cleavage of critical host cell proteins, acting to selectively 'hijack' host factors involved in gene expression, promoting picornavirus replication, and inactivating key factors in innate immunity signaling pathways. The protease and RNA-binding activities of 3C are involved in viral polyprotein processing and the initiation of viral RNA synthesis. Most importantly, 3C modifies critical molecules in host organelles and maintains virus infection by subtly subverting host cell death through the blocking of transcription, translation, and nucleocytoplasmic trafficking to modulate cell physiology for viral replication. Here, we discuss the molecular mechanisms through which 3C mediates physiological processes involved in promoting virus infection, replication, and release.

A Legionella toxin exhibits tRNA mimicry and glycosyl transferase activity to target the translation machinery and trigger a ribotoxic stress response.

A widespread strategy employed by pathogens to establish infection is to inhibit host-cell protein synthesis. Legionella pneumophila, an intracellular bacterial pathogen and the causative organism of Legionnaires' disease, secretes a subset of protein effectors into host cells that inhibit translation elongation. Mechanistic insights into how the bacterium targets translation elongation remain poorly defined. We report here that the Legionella effector SidI functions in an unprecedented way as a transfer-RNA mimic that directly binds to and glycosylates the ribosome. The 3.1 Å cryo-electron microscopy structure of SidI reveals an N-terminal domain with an 'inverted L' shape and surface-charge distribution characteristic of tRNA mimicry, and a C-terminal domain that adopts a glycosyl transferase fold that licenses SidI to utilize GDP-mannose as a sugar precursor. This coupling of tRNA mimicry and enzymatic action endows SidI with the ability to block protein synthesis with a potency comparable to ricin, one of the most powerful toxins known. In Legionella-infected cells, the translational pausing activated by SidI elicits a stress response signature mimicking the ribotoxic stress response, which is activated by elongation inhibitors that induce ribosome collisions. SidI-mediated effects on the ribosome activate the stress kinases ZAKα and p38, which in turn drive an accumulation of the protein activating transcription factor 3 (ATF3). Intriguingly, ATF3 escapes the translation block imposed by SidI, translocates to the nucleus and orchestrates the transcription of stress-inducible genes that promote cell death, revealing a major role for ATF3 in the response to collided ribosome stress. Together, our findings elucidate a novel mechanism by which a pathogenic bacterium employs tRNA mimicry to hijack a ribosome-to-nuclear signalling pathway that regulates cell fate.

A Dynamic and Static Binary Translation Method Based on Branch Prediction

Binary translation is an important technique for achieving cross-architecture software migration. However, mainstream dynamic binary translation frameworks, such as QEMU, often generate a large amount of redundant code, which degrades the efficiency of the target code. To this end, we propose a dynamic–static binary translation method based on branch prediction. It first identifies parts of translation blocks following static branch prediction techniques. Then it translates these translation blocks into less-redundant native code blocks by canonical static translation algorithms. Finally, it executes all code blocks that are translated either statically or dynamically by correctly maintaining and switching their running contexts. In order to correctly weave the two types of translation activities, the proposed method only translates the next translation block that is data-independent from the current one by the active variable analysis algorithm, and records and shares the intermediate states of the dynamic and static translation activities via a carefully designed data structure. In particular, a shadow register-based context recovery mechanism is proposed to correctly record the running context of static translation blocks, and to correctly recover the context for dynamically translating and running blocks that were not statically translated. We also designed an adaptive memory optimization mechanism to dynamically release the memory of the mispredicted translation blocks. We implemented a dynamic–static binary translation framework by extending QEMU, called BP-QEMU (QEMU with branch prediction). We evaluated the translation correctness of BP-QEMU using the testing programs for the ARM and PPC instruction sets from QEMU, and evaluated the performance of BP-QEMU using the CoreMark benchmark code. The experimental results show that BP-QEMU can translate the instructions from the ARM and PPC architectures correctly; moreover, the average execution efficiency of the CoreMark code on BP-QEMU improves by 13.3% compared to that of QEMU.

Regulation of TRIB1 abundance in hepatocyte models in response to proteasome inhibition

Tribbles related homolog 1 (TRIB1) contributes to lipid and glucose homeostasis by facilitating the degradation of cognate cargos by the proteasome. In view of the key metabolic role of TRIB1 and the impact of proteasome inhibition on hepatic function, we continue our exploration of TRIB1 regulation in two commonly used human hepatocyte models, transformed cell lines HuH-7 and HepG2. In both models, proteasome inhibitors potently upregulated both endogenous and recombinant TRIB1 mRNA and protein levels. Increased transcript abundance was unaffected by MAPK inhibitors while ER stress was a weaker inducer. Suppressing proteasome function via PSMB3 silencing was sufficient to increase TRIB1 mRNA expression. ATF3 was required to sustain basal TRIB1 expression and support maximal induction. Despite increasing TRIB1 protein abundance and stabilizing bulk ubiquitylation, proteasome inhibition delayed but did not prevent TRIB1 loss upon translation block. Immunoprecipitation experiments indicated that TRIB1 was not ubiquitylated in response to proteasome inhibition. A control bona fide proteasome substrate revealed that high doses of proteasome inhibitors resulted in incomplete proteasome inhibition. Cytoplasm retained TRIB1 was unstable, suggesting that TRIB1 lability is regulated prior to its nuclear import. N-terminal deletion and substitutions were insufficient to stabilize TRIB1. These findings identify transcriptional regulation as a prominent mechanism increasing TRIB1 abundance in transformed hepatocyte cell lines in response to proteasome inhibition and provide evidence of an inhibitor resistant proteasome activity responsible for TRIB1 degradation.

APOBEC3B drives PKR-mediated translation shutdown and protects stress granules in response to viral infection

Double-stranded RNA produced during viral replication and transcription activates both protein kinase R (PKR) and ribonuclease L (RNase L), which limits viral gene expression and replication through host shutoff of translation. In this study, we find that APOBEC3B forms a complex with PABPC1 to stimulate PKR and counterbalances the PKR-suppressing activity of ADAR1 in response to infection by many types of viruses. This leads to translational blockage and the formation of stress granules. Furthermore, we show that APOBEC3B localizes to stress granules through the interaction with PABPC1. APOBEC3B facilitates the formation of protein-RNA condensates with stress granule assembly factor (G3BP1) by protecting mRNA associated with stress granules from RNAse L-induced RNA cleavage during viral infection. These results not only reveal that APOBEC3B is a key regulator of different steps of the innate immune response throughout viral infection but also highlight an alternative mechanism by which APOBEC3B can impact virus replication without editing viral genomes.

MCP-1 facilitates VEGF production by removing miR-374b-5p blocking of VEGF mRNA translation

Monocyte chemotactic protein-1 (MCP-1) is known to be able to facilitate vascular endothelial growth factor (VEGF) gene expression, hence promoting vascular hyperpermeability and neovascularization. We show here that a microRNA molecule, miR-374b-5p can target the 3′-untranslated region of the VEGF mRNA, thus preventing VEGF production. Additionally, MCP-1 promotes the acetylation of transcription factor stat3 at Lys685, which facilitates the formation of an ac-stat3-DNA methyltransferase-histone methyltransferase complex (ac-stat3/DNMT1/EZH2) that binds to the promoter of the miR-374b-5p gene. This results in diminished miR-374b-5p expression and enhanced VEGF production. Moreover, treatment of appropriate animal models either with a miR-374b-5p mimicry or with inhibitors of either stat3 acetylation, DNMT1, or EZH2, leads to marked inhibition of MCP-1-promoted neovascularization and tumor growth. These findings indicate that MCP-1 facilitated inhibition of miR-374b-5p gene expression leads to the removal of a block of VEGF mRNA translation by miR-374b-5p. This mechanism could be of importance in the modulation of inflammatory conditions.

Design and Microinjection of Morpholino Antisense Oligonucleotides and mRNA into Zebrafish Embryos to Elucidate Specific Gene Function in Heart Development.

The morpholino oligomer-based knockdown system has been used to identify the function of various gene products through loss or reduced expression. Morpholinos (MOs) have the advantage in biological stability over DNA oligos because they are not susceptible to enzymatic degradation. For optimal effectiveness, MOs are injected into 1-4 cell stage embryos. The temporal efficacy of knockdown is variable, but MOs are believed to lose their effects due to dilution eventually. Morpholino dilution and injection amount should be closely controlled to minimize the occurrence of off-target effects while maintaining on-target efficacy. Additional complementary tools, such as CRISPR/Cas9 should be performed against the target gene of interest to generate mutant lines and to confirm the morphant phenotype with these lines. This article will demonstrate how to design, prepare, and microinject a translation-blocking morpholino against hand2 into the yolk of 1-4 cell stage zebrafish embryos to knockdown hand2 function and rescue these "morphants" by co-injection of mRNA encoding the corresponding cDNA. Subsequently, the efficacy of the morpholino microinjections is assessed by first verifying the presence of morpholino in the yolk (co-injected with phenol red) and then by phenotypic analysis. Moreover, cardiac functional analysis to test for knockdown efficacy will be discussed. Finally, assessing the effect of morpholino-induced blockage of gene translation via western blotting will be explained.

In vivo investigation of ruminant placenta function and physiology-a review.

The placenta facilitates the transport of nutrients to the fetus, removal of waste products from the fetus, immune protection of the fetus and functions as an endocrine organ, thereby determining the environment for fetal growth and development. Additionally, the placenta is a highly metabolic organ in itself, utilizing a majority of the oxygen and glucose derived from maternal circulation. Consequently, optimal placental function is required for the offspring to reach its genetic potential in utero. Among ruminants, pregnant sheep have been used extensively for investigating pregnancy physiology, in part due to the ability to place indwelling catheters within both maternal and fetal vessels, allowing for steady-state investigation of blood flow, nutrient uptakes and utilization, and hormone secretion, under non-stressed and non-anesthetized conditions. This methodology has been applied to both normal and compromised pregnancies. As such, our understanding of the in vivo physiology of pregnancy in sheep is unrivalled by any other species. However, until recently, a significant deficit existed in determining the specific function or significance of individual genes expressed by the placenta in ruminants. To that end, we developed and have been using in vivo RNA interference (RNAi) within the sheep placenta to examine the function and relative importance of genes involved in conceptus development (PRR15 and LIN28), placental nutrient transport (SLC2A1 and SLC2A3), and placenta-derived hormones (CSH). A lentiviral vector is used to generate virus that is stably integrated into the infected cell's genome, thereby expressing a short-hairpin RNA (shRNA), that when processed within the cell, combines with the RNA Induced Silencing Complex (RISC) resulting in specific mRNA degradation or translational blockage. To accomplish in vivo RNAi, day 9 hatched and fully expanded blastocysts are infected with the lentivirus for 4 to 5h, and then surgically transferred to synchronized recipient uteri. Only the trophectoderm cells are infected by the replication deficient virus, leaving the inner cell mass unaltered, and we often obtain ~70% pregnancy rates following transfer of a single blastocyst. In vivo RNAi coupled with steady-state study of blood flow and nutrient uptake, transfer and utilization can now provide new insight into the physiological consequences of modifying the translation of specific genes expressed within the ruminant placenta.

Role of Non-Coding RNA of Human Platelet in Cardiovascular Disease.

Cardiovascular diseases (CVD) are the major cause of death in the world. Numerous genetic studies involving transcriptomic approaches aimed at the detailed understanding of the disease and the development of new therapeutic strategies have been conducted over recent years. There has been an increase in research on platelets, which are implicated in CVD due to their capacity to release regulatory molecules that affect various pathways. Platelets secrete over 500 various kinds of molecules to plasma including large amounts of non-coding (nc) RNA (miRNA, lncRNA or circRNA). These ncRNA correspond to 98% of transcripts that are not translated into proteins as they are important regulators in physiology and disease. Thus, miRNAs can direct protein complexes to mRNAs through base-pairing interactions, thus causing translation blockage or/and transcript degradation. The lncRNAs act via different mechanisms by binding to transcription factors. Finally, circRNAs act as regulators of miRNAs, interfering with their action. Alteration in the repertoire and/or the amount of the platelet-secreted ncRNA can trigger CVD as well as other diseases. NcRNAs can serve as effective biomarkers for the disease or as therapeutic targets due to their disease involvement. In this review, we will focus on the most important ncRNAs that are secreted by platelets (9 miRNA, 9 lncRNA and 5 circRNA), their association with CVD, and the contribution of these ncRNA to CVD risk to better understand the relation between ncRNA of human platelet and CVD.

Regulation of autophagy by miRNAs in human diseases.

Autophagy is a homeostatic process designed to eliminate dysfunctional and aging organelles and misfolded proteins through a well-concerted pathway, starting with forming a double-membrane vesicle and culminating in the lysosomal degradation of the cargo enclosed inside the mature vesicle. As a vital sentry of cellular health, autophagy is regulated in every human disease condition and is an essential target for non-coding RNAs like microRNAs (miRNAs). miRNAs are short oligonucleotides that specifically bind to the 3'-untranslated region (UTR) of target mRNAs, thus leading to mRNA silencing, degradation, or translation blockage. This review summarizes the recent findings regarding the regulation of autophagy and autophagy-related genes by different miRNAs in various pathological conditions, including cancer, kidney and liver disorders, neurodegeneration, cardiovascular disorders, infectious diseases, aging-related conditions, and inflammation-related diseases. As miRNAs are being identified as prime regulators of autophagy in human disease, pharmacological molecules and traditional medicines targeting these miRNAs are also being tested in disease models, thus initiating a new series of therapeutic interventions targeting autophagy.

264 Using in vivo RNA Interference to Investigate Ruminant Placental Function

Abstract Pregnant sheep have been used extensively for investigating pregnancy physiology, providing valuable information about the progression of ruminant pregnancy. The ability to place indwelling catheters, within both maternal and fetal vessels, allows for steady-state investigation of blood flow, nutrient uptakes and utilization, and hormone secretion, under non-stressed and non-anesthetized conditions. As such, our understanding of the in vivo physiology of pregnancy in sheep is unrivalled by any other species. However, until recently, a significant deficit existed in determining the specific function or significance of individual genes expressed by the placenta in livestock. To that end, we developed and have been using in vivo RNA interference (RNAi) within the sheep placenta to examine the function and relative importance of genes involved in conceptus development (PRR15 and LIN28), placental nutrient transport (SLC2A1 and SLC2A3), and placenta derived hormones (CSH). The lentiviral vector LL3.7 is used to generate virus that is stably integrated into the infected cell’s genome, thereby expressing a short-hairpin RNA (shRNA), that when processed within the cell, combines with the RNA Induced Silencing Complex (RISC) resulting in specific mRNA degradation or translational blockage. To accomplish in vivo RNAi, day 9 hatched and fully expanded blastocysts are infected with the lentivirus for 4–5 hours, and then surgically transferred to synchronized recipient uteri. Only the trophectoderm cells are infected by the replication deficient virus, leaving the inner cell mass unaltered, and we typically obtain 70–80% pregnancy rates following transfer of a single blastocyst. Data will be presented from two projects. One is focused on generating a deficiency in placental glucose transporters at mid-gestation, and the other on the impact of CSH RNAi during late gestation, demonstrating the utility of this experimental approach for examining gene function within the placenta of livestock. Supported by NIH-NICHD grants HD093701 and HD094952.

Stroke recovery enhancing therapies: lessons from recent clinical trials.

Poststroke recovery processes include restoration or compensation of function, respectively functions initially lost or new functions acquired after an injury. Therapeutic interventions can enhance these processes and/or reduce processes impeding regeneration. Numerous experimental studies suggest great opportunities for such treatments, but the results from recent large clinical trials using neuromodulators such as dopamine and fluoxetine are disappointing. The reasons for this are manifold affecting forward translation of results from animals models into the human situation. This “translational road block” is defined by differences between animals and humans with regard to the genetic and epigenetic background, size and anatomy of the brain, cerebral vascular anatomy, immune system, as well as clinical function and behavior. Backward blockade includes the incompatible adaption of targets and outcomes in clinical trials with regard to prior preclinical findings. For example, the design of clinical recovery trials varies widely and was characterized by the selection of different clinical endpoints, the inclusion a broad spectrum of stroke subtypes and clinical syndromes as well as different time windows for treatment initiation after infarct onset. This review will discuss these aspects based on the results of the recent stroke recovery trials with the goal to contribute to the currently biggest unmet need in stroke research - the development of a recovery enhancing therapy that improves the functional outcome of a chronic stroke patient.