Genetic Intervention Strategies Research Articles

ABC transporters mined through comparative transcriptomics associate with organ-specific accumulation of picrosides in a medicinal herb, Picrorhiza kurroa.

Picrorhiza kurroa Royle ex Benth is a valuable medicinal herb of North-Western Himalayas due to presence of two major bioactive compounds, picroside-I and picroside-II used in the preparation of several hepatoprotective herbal drugs. These compounds accumulate in stolons/rhizomes; however, biosynthesized in different organs, viz., picroside-I in shoots and picroside-II in roots. As of today, no information exists on what transporters are transporting these metabolites from shoots and roots to the final storage organ, stolon, which ultimately transforms into rhizome. The ATP-binding cassette (ABC) transporters are reported to transport majority of secondary metabolites, including terpenoids in plants, therefore, we mined P. kurroa transcriptomes to identify and shortlist potential candidates. A total of 99 ABC transporter-encoding transcripts were identified in 3 differential transcriptomes, PKSS (shoots), PKSTS (stolons), and PKSR (roots) of P. kurroa, based on in silico comparative analysis and transcript abundance. 15 of these transcripts were further validated for their association using qRT-PCR in shoots, roots and stolon tissues in P. kurroa accessions varying for picroside-I and picroside-II contents. Organ-specific expression analysis revealed that PkABCA1, PkABCG1, and PkABCB5 had comparatively elevated expression in shoots; PkABCB2 and PkABCC2 in roots; PkABCB3 and PkABCC1 in stolon tissues of P. kurroa. Co-expression network analysis using ABC genes as hubs further unravelled important interactions with additional components of biosynthetic machinery. Our study has provided leads, first to our knowledge as of today, on putative ABC transporters possibly involved in long distance and local transport of picrosides in P. kurroa organs, thus opening avenues for designing a suitable genetic intervention strategy.

A Genome-Scale Metabolic Model of Anabaena 33047 to Guide Genetic Modifications to Overproduce Nylon Monomers.

Nitrogen fixing-cyanobacteria can significantly improve the economic feasibility of cyanobacterial production processes by eliminating the requirement for reduced nitrogen. Anabaena sp. ATCC 33047 is a marine, heterocyst forming, nitrogen fixing cyanobacteria with a very short doubling time of 3.8 h. We developed a comprehensive genome-scale metabolic (GSM) model, iAnC892, for this organism using annotations and content obtained from multiple databases. iAnC892 describes both the vegetative and heterocyst cell types found in the filaments of Anabaena sp. ATCC 33047. iAnC892 includes 953 unique reactions and accounts for the annotation of 892 genes. Comparison of iAnC892 reaction content with the GSM of Anabaena sp. PCC 7120 revealed that there are 109 reactions including uptake hydrogenase, pyruvate decarboxylase, and pyruvate-formate lyase unique to iAnC892. iAnC892 enabled the analysis of energy production pathways in the heterocyst by allowing the cell specific deactivation of light dependent electron transport chain and glucose-6-phosphate metabolizing pathways. The analysis revealed the importance of light dependent electron transport in generating ATP and NADPH at the required ratio for optimal N2 fixation. When used alongside the strain design algorithm, OptForce, iAnC892 recapitulated several of the experimentally successful genetic intervention strategies that over produced valerolactam and caprolactam precursors.

Identification of Candidate Signature Genes and Key Regulators Associated With Trypanotolerance in the Sheko Breed.

African animal trypanosomiasis (AAT) is caused by a protozoan parasite that affects the health of livestock. Livestock production in Ethiopia is severely hampered by AAT and various controlling measures were not successful to eradicate the disease. AAT affects the indigenous breeds in varying degrees. However, the Sheko breed shows better trypanotolerance than other breeds. The tolerance attributes of Sheko are believed to be associated with its taurine genetic background but the genetic controls of these tolerance attributes of Sheko are not well understood. In order to investigate the level of taurine background in the genome, we compare the genome of Sheko with that of 11 other African breeds. We find that Sheko has an admixed genome composed of taurine and indicine ancestries. We apply three methods: (i) The integrated haplotype score (iHS), (ii) the standardized log ratio of integrated site specific extended haplotype homozygosity between populations (Rsb), and (iii) the composite likelihood ratio (CLR) method to discover selective sweeps in the Sheko genome. We identify 99 genomic regions harboring 364 signature genes in Sheko. Out of the signature genes, 15 genes are selected based on their biological importance described in the literature. We also identify 13 overrepresented pathways and 10 master regulators in Sheko using the TRANSPATH database in the geneXplain platform. Most of the pathways are related with oxidative stress responses indicating a possible selection response against the induction of oxidative stress following trypanosomiasis infection in Sheko. Furthermore, we present for the first time the importance of master regulators involved in trypanotolerance not only for the Sheko breed but also in the context of cattle genomics. Our finding shows that the master regulator Caspase is a key protease which plays a major role for the emergence of adaptive immunity in harmony with the other master regulators. These results suggest that designing and implementing genetic intervention strategies is necessary to improve the performance of susceptible animals. Moreover, the master regulatory analysis suggests potential candidate therapeutic targets for the development of new drugs for trypanosomiasis treatment.

MoVE identifies metabolic valves to switch between phenotypic states

Metabolism is highly regulated, allowing for robust and complex behavior. This behavior can often be achieved by controlling a small number of important metabolic reactions, or metabolic valves. Here, we present a method to identify the location of such valves: the metabolic valve enumerator (MoVE). MoVE uses a metabolic model to identify genetic intervention strategies which decouple two desired phenotypes. We apply this method to identify valves which can decouple growth and production to systematically improve the rate and yield of biochemical production processes. We apply this algorithm to the production of diverse compounds and obtained solutions for over 70% of our targets, identifying a small number of highly represented valves to achieve near maximal growth and production. MoVE offers a systematic approach to identify metabolic valves using metabolic models, providing insight into the architecture of metabolic networks and accelerating the widespread implementation of dynamic flux redirection in diverse systems.

Comparative transcriptomics reveals a reduction in carbon capture and flux between source and sink in cytokinin-treated inflorescences of Jatropha curcas L.

The low seed yield of Jatropha curcas has been a stumbling block in realizing its full potential as an ideal bioenergy crop. Low female to male flower ratio is considered as a major limiting factor responsible for low seed yield in Jatropha. An exogenous cytokinin application was performed on floral meristems to increase the seed yield. This resulted in an increase of total flowers count with a higher female to male flower ratio. However, the seed biomass did not increase in the same proportion. The possible reason for this was hypothesized to be the lack of increased photosynthesis efficiency at source tissues which could fulfil the increased demand of photosynthates and primary metabolites in maturing seeds. After cytokinin application, possible molecular mechanisms underlying carbon capture and flux affected between the source and sink in developing flowers, fruits and seeds were investigated. Comparative transcriptome analysis was performed on inflorescence meristems (treated with cytokinin) and control (untreated inflorescence meristems) at time intervals of 15 and 30days, respectively. KEGG-based functional annotation identified various metabolic pathways associated with carbon capture and flux. Pathways such as photosynthesis, carbon fixation, carbohydrate metabolism and nitrogen metabolism were upregulated after 15days of cytokinin treatment; however, those were downregulated after 30days. Five genes FBP, SBP, GS, GDH and AGPase showed significant increase in transcript abundance after 15days of treatment but showed a significant decrease after 30days. These genes, after functional validation, can be suitable targets in designing a suitable genetic intervention strategy to increase overall seed yield in Jatropha.

Designing Optimized Production Hosts by Metabolic Modeling.

Many of the complex and expensive production steps in the chemical industry are readily available in living cells. In order to overcome the metabolic limits of these cells, the optimal genetic intervention strategies can be computed by the use of metabolic modeling. Elementary flux mode analysis (EFMA) is an ideal tool for this task, as it does not require defining a cellular objective function. We present two EFMA-based methods to optimize production hosts: (1) the standard approach that can only be used for small and medium scale metabolic networks and (2) the advanced dual system approach that can be utilized to directly compute intervention strategies in a genome-scale metabolic model.

Transcriptomes of Podophyllum hexandrum unravel candidate miRNAs and their association with the biosynthesis of secondary metabolites

Podophyllotoxin is a high value metabolite having anticancerous activity and procured from a medicinal plant Podophyllum hexandrum. The escalating demand for podophyllotoxin necessitates development of alternate production platforms. Cell cultures have been tried in the past, however, podophyllotoxin yields were very low (0.3%). Multiple genes of biosynthetic pathway have been correlated with podophyllotoxin content. Understanding regulation of podophyllotoxin is, therefore, essential to design a suitable genetic intervention strategy. Six potential miRNAs were identified in NGS transcriptomes of P. hexandrum for which eight potential mRNA targets were discerned. The identified miRNAs and their mRNA targets (UDP glycosyltransferase, flavonol synthase, glyceraldehyde 3-phosphate dehydrogenase, peroxidase, malate dehydrogenase, phosphoenolpyruvate carboxylase, WRKY 37 and MYBF1 transcription factors) were further tested for any correlation through qRT-PCR. miR396b, miR2673a, miR828b and miR2910 established negative correlation with their mRNA targets through downregulation via complementary miRNA:mRNA base pairing. This is the first report on identification, characterisation, validation and expression analysis of miRNAs and their targets in P. hexandrum.

Mathematical modeling of erythrocyte chimerism informs genetic intervention strategies for sickle cell disease.

Recent advances in gene therapy and genome-engineering technologies offer the opportunity to correct sickle cell disease (SCD), a heritable disorder caused by a point mutation in the β-globin gene. The developmental switch from fetal γ-globin to adult β-globin is governed in part by the transcription factor (TF) BCL11A. This TF has been proposed as a therapeutic target for reactivation of γ-globin and concomitant reduction of β-sickle globin. In this and other approaches, genetic alteration of a portion of the hematopoietic stem cell (HSC) compartment leads to a mixture of sickling and corrected red blood cells (RBCs) in periphery. To reverse the sickling phenotype, a certain proportion of corrected RBCs is necessary; the degree of HSC alteration required to achieve a desired fraction of corrected RBCs remains unknown. To address this issue, we developed a mathematical model describing aging and survival of sickle-susceptible and normal RBCs; the former can have a selective survival advantage leading to their overrepresentation. We identified the level of bone marrow chimerism required for successful stem cell-based gene therapies in SCD. Our findings were further informed using an experimental mouse model, where we transplanted mixtures of Berkeley SCD and normal murine bone marrow cells to establish chimeric grafts in murine hosts. Our integrative theoretical and experimental approach identifies the target frequency of HSC alterations required for effective treatment of sickling syndromes in humans. Our work replaces episodic observations of such target frequencies with a mathematical modeling framework that covers a large and continuous spectrum of chimerism conditions. Am. J. Hematol. 91:931-937, 2016. © 2016 Wiley Periodicals, Inc.

Prospecting NGS-transcriptomes to assess regulation of miRNA-mediated secondary metabolites biosynthesis in Swertia chirayita, a medicinal herb of the North-Western Himalayas

Swertia chirayita is a high value medicinal herb that possesses diverse therapeutic traits. Molecular data related to the biosynthesis of major secondary metabolites exhibiting these pharmacological properties are limited. Growing demand and poor cultural procedures necessitate improved genetic intervention strategies, which can be implemented to positively influence the biosynthetic machinery and its regulatory network. miRNAs are indicated to play pivotal role in various biological processes, including growth and secondary metabolism. In an attempt to identify conserved miRNAs, computational methods were followed across two transciptomes of Swertia contrasting for metabolite levels and cultural conditions. A total of ten miRNAs were predicted followed by target prediction that resulted in the identification of eleven potential targets having roles in secondary metabolism. RT-qPCR analysis showed six miRNAs (miR-168, miR-11320, miR-166a, miR-11071, miR-156a and miR-166b), targeting Acetyl -CoA acetyltransferase (AACT), Aspar tate aminotransferase (PHAT), premnaspirodiene oxygenase (PSO), ribulose-phosphate 3-epimerase (RPE), phosphoglycerate mutase (PGM) and a gene encoding homeobox-leucine zipper protein (HD-ZIP) having negative correlations with their targets, thereby indicating their possible association in secondary metabolites biosynthesis. The study sheds light on miRNA mediated regulation of secondary metabolites biosynthesis in a lesser explored herbal plant Swertia chirayita. Besides, the putatively identified miRNAs can be targeted for genetic manipulation to enhance secondary metabolites biosynthesis in S. chirayita.

NGS Transcriptomes and Enzyme Inhibitors Unravel Complexity of Picrosides Biosynthesis in Picrorhiza kurroa Royle ex. Benth

Picrorhiza kurroa is an important medicinal herb valued for iridoid glycosides, Picroside-I (P-I) and Picroside-II (P-II), which have several pharmacological activities. Genetic interventions for developing a picroside production platform would require knowledge on biosynthetic pathway and key control points, which does not exist as of today. The current study reports that geranyl pyrophosphate (GPP) moiety is mainly contributed by the non-mevalonate (MEP) route, which is further modified to P-I and P-II through phenylpropanoid and iridoid pathways, in total consisting of 41 and 35 enzymatic steps, respectively. The role of the MEP pathway was ascertained through enzyme inhibitors fosmidomycin and mevinolin along with importance of other integrating pathways using glyphosate, aminooxy acetic acid (AOA) and actinomycin D, which overall resulted in 17%-92% inhibition of P-I accumulation. Retrieval of gene sequences for enzymatic steps from NGS transcriptomes and their expression analysis vis-à-vis picrosides content in different tissues/organs showed elevated transcripts for twenty genes, which were further shortlisted to seven key genes, ISPD, DXPS, ISPE, PMK, 2HFD, EPSPS and SK, on the basis of expression analysis between high versus low picrosides content strains of P. kurroa so as to eliminate tissue type/ developmental variations in picrosides contents. The higher expression of the majority of the MEP pathway genes (ISPD, DXPS and ISPE), coupled with higher inhibition of DXPR enzyme by fosmidomycin, suggested that the MEP route contributed to the biosynthesis of P-I in P. kurroa. The outcome of the study is expected to be useful in designing a suitable genetic intervention strategy towards enhanced production of picrosides. Possible key genes contributing to picroside biosynthesis have been identified with potential implications in molecular breeding and metabolic engineering of P. kurroa.

Lipid content in Scenedesmus species correlates with multiple genes of fatty acid and triacylglycerol biosynthetic pathways

The feasibility of microalgae based biodiesel depends upon the selection of strains having high lipid content without compromising growth. The molecular basis governing storage lipid biosynthesis is poorly understood in microalgae including Scenedesmus, which has been identified as a potential oil-producing microalga. Eighteen genes of fatty acid and triacylglycerol biosynthesis were identified in Scenedesmus dimorphus through comparative genomics and transcriptome mining and their expression was examined in three growth stages viz., exponential, early stationary, late stationary phase and two stress conditions induced by nitrogen deficiency and high salinity among two lipid contrasting strains of S. dimorphus, SD12 (26%), SD16 (5%) and one strain of Scenedesmus quadricauda SQ19 (14%). Most of the genes showed relatively higher expression in high lipid strain, SD12 with up to 50 folds and 25 folds increase in transcript abundance of 9 genes compared to SD16 and SQ19, respectively. Eleven genes showed upregulation at late stationary phase and under stress conditions, suggesting role of multiple genes in controlling lipid content. Comparison of gene expression across differential conditions among three contrasting strains unveiled six key genes, ACP, FATA, KASII, LPAAT, PAP and DGAT that can be chosen as potential targets for designing a genetic intervention strategy for further enrichment of lipid profile in Scenedesmus species.

Next-generation sequencing (NGS) transcriptomes reveal association of multiple genes and pathways contributing to secondary metabolites accumulation in tuberous roots of Aconitum heterophyllum Wall.

The transcriptomes of Aconitum heterophyllum were assembled and characterized for the first time to decipher molecular components contributing to biosynthesis and accumulation of metabolites in tuberous roots. Aconitum heterophyllum Wall., popularly known as Atis, is a high-value medicinal herb of North-Western Himalayas. No information exists as of today on genetic factors contributing to the biosynthesis of secondary metabolites accumulating in tuberous roots, thereby, limiting genetic interventions towards genetic improvement of A. heterophyllum. Illumina paired-end sequencing followed by de novo assembly yielded 75,548 transcripts for root transcriptome and 39,100 transcripts for shoot transcriptome with minimum length of 200 bp. Biological role analysis of root versus shoot transcriptomes assigned 27,596 and 16,604 root transcripts; 12,340 and 9398 shoot transcripts into gene ontology and clusters of orthologous group, respectively. KEGG pathway mapping assigned 37 and 31 transcripts onto starch-sucrose metabolism while 329 and 341 KEGG orthologies associated with transcripts were found to be involved in biosynthesis of various secondary metabolites for root and shoot transcriptomes, respectively. In silico expression profiling of the mevalonate/2-C-methyl-D-erythritol 4-phosphate (non-mevalonate) pathway genes for aconites biosynthesis revealed 4 genes HMGR (3-hydroxy-3-methylglutaryl-CoA reductase), MVK (mevalonate kinase), MVDD (mevalonate diphosphate decarboxylase) and HDS (1-hydroxy-2-methyl-2-(E)-butenyl 4-diphosphate synthase) with higher expression in root transcriptome compared to shoot transcriptome suggesting their key role in biosynthesis of aconite alkaloids. Five genes, GMPase (geranyl diphosphate mannose pyrophosphorylase), SHAGGY, RBX1 (RING-box protein 1), SRF receptor kinases and β-amylase, implicated in tuberous root formation in other plant species showed higher levels of expression in tuberous roots compared to shoots. A total of 15,487 transcription factors belonging to bHLH, MYB, bZIP families and 399 ABC transporters which regulate biosynthesis and accumulation of bioactive compounds were identified in root and shoot transcriptomes. The expression of 5 ABC transporters involved in tuberous root development was validated by quantitative PCR analysis. Network connectivity diagrams were drawn for starch-sucrose metabolism and isoquinoline alkaloid biosynthesis associated with tuberous root growth and secondary metabolism, respectively, in root transcriptome of A. heterophyllum. The current endeavor will be of practical importance in planning a suitable genetic intervention strategy for the improvement of A. heterophyllum.

Logical transformation of genome-scale metabolic models for gene level applications and analysis.

In recent years, genome-scale metabolic models (GEMs) have played important roles in areas like systems biology and bioinformatics. However, because of the complexity of gene-reaction associations, GEMs often have limitations in gene level analysis and related applications. Hence, the existing methods were mainly focused on applications and analysis of reactions and metabolites. Here, we propose a framework named logic transformation of model (LTM) that is able to simplify the gene-reaction associations and enables integration with other developed methods for gene level applications. We show that the transformed GEMs have increased reaction and metabolite number as well as degree of freedom in flux balance analysis, but the gene-reaction associations and the main features of flux distributions remain constant. In addition, we develop two methods, OptGeneKnock and FastGeneSL by combining LTM with previously developed reaction-based methods. We show that the FastGeneSL outperforms exhaustive search. Finally, we demonstrate the use of the developed methods in two different case studies. We could design fast genetic intervention strategies for targeted overproduction of biochemicals and identify double and triple synthetic lethal gene sets for inhibition of hepatocellular carcinoma tumor growth through the use of OptGeneKnock and FastGeneSL, respectively. Source code implemented in MATLAB, RAVEN toolbox and COBRA toolbox, is public available at https://sourceforge.net/projects/logictransformationofmodel.

Expression analysis of biosynthetic pathway genes vis-à-vis podophyllotoxin content in Podophyllum hexandrum Royle.

Podophyllum hexandrum Royle is known for its vast medicinal properties, particularly anticancer. It contains higher amount of podophyllotoxin (4.3 %), compared to Podophyllum peltatum (0.025 %) and other plant species; as a result, it has been used worldwide in the preparation of various drugs including anticancer, antimalarial, antiviral, antioxidant, antifungal, and so on. Currently, Etoposide (VP-16-213), Vumon® (Teniposide; VM-26), Etopophos®, Pod-Ben- 25, Condofil, Verrusol, and Warticon are available in the market. Due to highly complex synthesis and low cell culture yields of podophyllotoxin (0.3 %), the supply of raw material cannot be met due to increasing industrial demands. The knowledge on podophyllotoxin biosynthetic pathway vis-à-vis expression status of genes is fragmentary. Quantitative expression analysis of 21 pathway genes has revealed 9 genes, namely SD, PD, PCH, CM, CMT, CAD, CCR, C4H, and ADH, that showed increase in transcript abundance up to 1.4 to 23.05 folds, respectively, vis-à-vis podophyllotoxin content in roots (1.37 %) and rhizomes (3.05 %) of P. hexandrum. In silico analysis of putative cis-regulatory elements in promoter regions of overexpressed genes showed the presence of common Skn-1 motif and MBS elements in CMT, CAD, CCR, C4H, and ADH genes, thereby, suggesting their common regulation. The outcome of the study has resulted in the identification of suitable candidate genes which might be contributing to podophyllotoxin biosynthesis that can act as potential targets for any genetic intervention strategies aimed at its enhanced production.

CCR5 gene disruption via lentiviral vectors expressing Cas9 and single guided RNA renders cells resistant to HIV-1 infection.

CCR5, a coreceptor for HIV-1 entry, is a major target for drug and genetic intervention against HIV-1. Genetic intervention strategies have knocked down CCR5 expression levels by shRNA or disrupted the CCR5 gene using zinc finger nucleases (ZFN) or Transcription activator-like effector nuclease (TALEN). In the present study, we silenced CCR5 via CRISPR associated protein 9 (Cas9) and single guided RNAs (sgRNAs). We constructed lentiviral vectors expressing Cas9 and CCR5 sgRNAs. We show that a single round transduction of lentiviral vectors expressing Cas9 and CCR5 sgRNAs into HIV-1 susceptible human CD4+ cells yields high frequencies of CCR5 gene disruption. CCR5 gene-disrupted cells are not only resistant to R5-tropic HIV-1, including transmitted/founder (T/F) HIV-1 isolates, but also have selective advantage over CCR5 gene-undisrupted cells during R5-tropic HIV-1 infection. Importantly, using T7 endonuclease I assay we did not detect genome mutations at potential off-target sites that are highly homologous to these CCR5 sgRNAs in stably transduced cells even at 84 days post transduction. Thus we conclude that silencing of CCR5 via Cas9 and CCR5-specific sgRNAs could be a viable alternative strategy for engineering resistance against HIV-1.

Quantitative analysis of the green microalga Coccomyxa subellipsoidea C169 reveals homeostasis of complex lipid synthesis retained during nitrogen stress (605.14)

The inevitable decline of fossil fuel availability and the destructive escalation of atmospheric carbon dioxide concentration are pressing problems that scientific innovation can potentially address. Algae accumulate large amounts of oil when exposed to abiotic stress, enough to possibility use their cultivation to produce carbon‐neutral replacements of conventional transportation fuels. Unraveling the intricacies of triglyceride (TG) synthesis during abiotic stress is a means to generate genetic intervention strategies for improving lipid productivity. Our research is focused on understanding TG accumulation in the context of lipid biosynthesis for Coccomyxa subellipsoidea C169, a sequenced relative of Chlorella and a sensible proxy for biofuel production species. A thorough analysis of lipid synthesis was conducted including GC‐MS fatty acid profiles of isolated lipid pools and LC‐MS/MS quantification of membrane lipid (ML) and TG pools through a time course of N starvation. Stable isotopic labeling studies were done with 13CO2 and D2O to analyze the roles of photosynthesis and de novo synthesis from previously reduced carbon in TG synthesis. The data quantify an initial redistribution of membrane lipids into the TG pool followed by synthesis and incorporation of fatty acids into all complex lipid pools. Reproductive growth is inhibited by N stress, however lipid synthesis continues normally.Grant Funding Source: Supported by NSF‐EPSCoR, Nebraska Center for Algal Biology and Biotechnology

Comparison and improvement of algorithms for computing minimal cut sets

BackgroundConstrained minimal cut sets (cMCSs) have recently been introduced as a framework to enumerate minimal genetic intervention strategies for targeted optimization of metabolic networks. Two different algorithmic schemes (adapted Berge algorithm and binary integer programming) have been proposed to compute cMCSs from elementary modes. However, in their original formulation both algorithms are not fully comparable.ResultsHere we show that by a small extension to the integer program both methods become equivalent. Furthermore, based on well-known preprocessing procedures for integer programming we present efficient preprocessing steps which can be used for both algorithms. We then benchmark the numerical performance of the algorithms in several realistic medium-scale metabolic models. The benchmark calculations reveal (i) that these preprocessing steps can lead to an enormous speed-up under both algorithms, and (ii) that the adapted Berge algorithm outperforms the binary integer approach.ConclusionsGenerally, both of our new implementations are by at least one order of magnitude faster than other currently available implementations.

GDNF Gene Delivery via a 2-(Dimethylamino)ethyl Methacrylate Based Cyclized Knot Polymer for Neuronal Cell Applications

Nonviral genetic therapeutic intervention strategies for neurological disorders hold great promise, but a lack of vector efficacy, coupled with vector toxicity, continue to hinder progress. Here we report the application of a newly developed class of polymer, distinctly different from conventional branched polymers, as a transfection agent for the delivery of glial cell line derived neurotrophic factor (GDNF) encoding gene. This new 2-(dimethylamino)ethyl methacrylate (DMAEMA) based cyclized knot polymer was studied for neuronal cell transfection applications, in comparison to branched polyethyleneimine (PEI). While showing a similar transfection profile over multiple cell types, the cyclized knot polymer showed far lower toxicity. In addition, transfection of Neu7 astrocytes with the GDNF encoding gene was able to cause neurite outgrowth when cocultured with dorsal root ganglia (DRGs). The cyclized knot polymer assessed here (PD-E 8%PEG), synthesized via a simple one-pot reaction, was shown to have great potential for neuronal gene therapy applications.

Therapeutic Targeting of IL-6 Trans Signaling Counteracts STAT3 Control of Experimental Inflammatory Arthritis

Cytokine control of the synovial infiltrate is a central process in the development of inflammatory arthritis. In this study, we combine genetic approaches and intervention strategies to describe a fundamental requirement for IL-6-mediated STAT3 signaling in orchestrating the inflammatory infiltrate in monoarticular and systemic models of experimental arthritis. STAT3 activation via the common gp130 signal-transducing receptor for all IL-6-related cytokines led to increased retention of neutrophils and T cells within the inflamed synovium, which included STAT3-regulated IL-17A-secreting T cells. Control of leukocyte infiltration was reliant upon IL-6 signaling via its soluble receptor (termed IL-6 trans signaling), as evidenced by selective blockade of this alternative IL-6 signaling pathway using an engineered variant of soluble gp130 (sgp130Fc). This therapeutic intervention led to substantial clinical improvement in mice with emerging or established incidence of systemic arthritis. These data illustrate that IL-6 control of STAT3 is critical for regulating the synovial infiltrate in inflammatory arthritis, and suggest that selective inhibition of IL-6 trans signaling may provide a more refined intervention strategy for blocking IL-6-driven proarthritic activities.

Grid cells in mice

The medial entorhinal cortex (EC) is a part of the neural network for the representation of self-location in the rat. The key cell type of this system is the grid cell, whose multiple firing fields span the environment in a remarkably regular triangular or hexagonal pattern. The basic properties of grid cells and other cell types have been described, but the neuronal mechanisms responsible for the formation and maintenance of the place code remain elusive. These mechanisms can be investigated by genetic intervention strategies, where specific components of the entorhinal-hippocampal network are activated or silenced. Because of the common use of knockout mice for such targeted interventions, we asked if grid activity is expressed also in the mouse. Principal neurons in the superficial layers of mouse medial EC had stable grid fields similar to those of the rat. Neighboring grid cells shared a common spacing and orientation but had a different spatial phase, such that a small number of grid cells collectively represented all locations in the environment. The spacing of the grid increased with distance from the dorsal border of the medial EC. The lowest values for grid spacing, recorded at the dorsal end, were comparable to those of the rat, suggesting that grid fields do not scale up proportionally with body size. Grid cells were colocalized with head-direction cells and conjunctive place x head-direction cells, as in the rat. The demonstration of grid cells in mice prepares the ground for transgenic analyses of the entorhinal-hippocampal network.