Gene Expression Localization Research Articles

Differential Expression of Local Immune Response Genes in the Vagina: Implication for the Diagnosis of Vaginal Infections.

Transcription profiles of genes of local immune response were determined in the vagina of women with bacterial vaginosis, aerobic vaginitis, and vulvovaginal candidosis for detection of the most specific immune markers for these vaginal infections. Laboratory diagnosis of the vaginal infections was performed microscopically; the inflammatory reaction in the vagina (leukorrhea) was defined as the presence of >10 white blood cells per field of view. Transcription profiles of IL1b, IL10, IL18, TNFα, TLR4, GATA3, and CD68 were determined using reverse-transcription quantitative real-time PCR. The strongest predictors of aerobic vaginitis were increased levels of IL1b and IL10 mRNA. Bacterial vaginosis was strongly associated with reduced levels of IL18 and GATA3 mRNA. Increased levels of IL1b and TLR4 transcripts showed significant discriminatory power for vulvovaginal candidosis and leukorrhea. The results of this study suggest differential expression of local immune response genes in the vagina of women with different vaginal infections. Detection of specific immune markers in the vagina using reverse-transcriptase PCR could supplement PCR detection of abnormal vaginal microflora for the diagnosis of vaginal infections.

A regulatory circuit between lncRNA and TOR directs amino acid uptake in yeast

Long non coding RNAs (lncRNAs) have emerged as crucial players of several central cellular processes across eukaryotes. Target of Rapamycin (TOR) is a central regulator of myriad of fundamental cellular processes including amino acid transport under diverse environmental conditions. Here we investigated the role of lncRNA in TOR regulated amino acid uptake in S. cerevisiae. Transcription of lncRNA regulates local gene expression in eukaryotes. In silico analysis of many genome wide studies in S. cerevisiae revealed that transcriptome includes conditional expression of numerous lncRNAs in proximity to amino acid transporters (AATs). Considering regulatory role of these lncRNAs, we selected highly conserved TOR regulated locus of a pair of AATs present in tandem BAP2 and TAT1. We observed that the expression of antisense lncRNA XUT_2F-154 (TBRT) and AATs BAP2 and TAT1 depends on activities of TOR signaling pathway. The expression of TBRT is induced, while that of BAP2 TAT1 is repressed upon TOR inhibition by Torin2. Notably, upon TOR inhibition loss of TBRT contributed to enhanced activities of Bap2 and Tat1 leading to improved growth. Interestingly, nucleosome scanning assay reveal that TOR signaling pathway governs chromatin remodeling at BAP2 biphasic promoter to control the antagonism of TBRT and BAP2 expression. Further TBRT also reprograms local chromatin landscapes to decrease the transcription of TAT1. The current work demonstrates a functional correlation between lncRNA production and TOR governed amino acid uptake in yeast. Thus this work brings forth a novel avenue for identification of potential regulators for therapeutic interventions against TOR mediated diseases.

Caterpillar Oral Secretion Elicits Reactive Oxygen Species in Isolated Plant Protoplasts

Over millions of years, plants have evolved and developed several defense strategies such as spines, trichomes, toxins, and plant volatiles to either directly or indirectly defend against insect herbivores. Reactive Oxygen Species (ROS) is a signaling molecule that is suspected of signaling herbivory by activating defense cascade at the cellular level, leading to local and systemic defense gene expression. In this study, we show that tobacco hornworm caterpillar (Manduca sexta) oral secretion (OS) induces ROS in tomato (Solanum lycopersicum) protoplasts. We used a dye-based ROS imaging approach to dissect the interaction between a damaging herbivore and its host plant. Our study shows that application of Plant-Fed (PF) caterpillar OS increased ROS generation while artificial Diet-Fed (DF) caterpillar OS failed to induce ROS in isolated tomato protoplasts. The elevation in ROS generation was observed after ˜100 s and was saturated after 165 s of PF-OS application. Interestingly, the PF-OS induced ROS increase was abolished in the presence of a Ca2+ chelator BAPTA-AM, suggesting possible crosstalk between Ca2+ and ROS signaling. Targeting the pathway that triggers ROS generation could be used to develop a herbivore origin potent elicitor of plant defense gene expression (ROS elicitor) pest management research.

Both Biosynthesis and Transport Are Involved in Glucosinolate Accumulation During Root-Herbivory in Brassica rapa.

The optimal defense theory predicts that plants invest most energy in those tissues that have the highest value, but are most vulnerable to attacks. In Brassica species, root-herbivory leads to the accumulation of glucosinolates (GSLs) in the taproot, the most valuable belowground plant organ. Accumulation of GSLs can result from local biosynthesis in response to herbivory. In addition, transport from distal tissues by specialized GSL transporter proteins can play a role as well. GSL biosynthesis and transport are both inducible, but the role these processes play in GSL accumulation during root-herbivory is not yet clear. To address this issue, we performed two time-series experiments to study the dynamics of transport and biosynthesis in local and distal tissues of Brassica rapa. We exposed roots of B. rapa to herbivory by the specialist root herbivore Delia radicum for 7 days. During this period, we sampled above- and belowground plant organs 12 h, 24 h, 3 days and 7 days after the start of herbivory. Next, we measured the quantity and composition of GSL profiles together with the expression of genes involved in GSL biosynthesis and transport. We found that both benzyl and indole GSLs accumulate in the taproot during root-herbivory, whereas we did not observe any changes in aliphatic GSL levels. The rise in indole GSL levels coincided with increased local expression of biosynthesis and transporter genes, which suggest that both biosynthesis and GSL transport play a role in the accumulation of GSLs during root herbivory. However, we did not observe a decrease in GSL levels in distal tissues. We therefore hypothesize that GSL transporters help to retain GSLs in the taproot during root-herbivory.

Different expression of Defensin-B gene in the endometrium of mares of different age during the breeding season

BackgroundDespite being one of the major causes of infertility in mares, the mechanisms responsible for equine endometrosis are still unclear and controversial. In the last few years, many investigations focused on local immune response modulation. Since it is generally accepted that endometrial fibrosis increases with age, we hypothesize that older mares could show altered local immune modulation, initiating a pro-inflammatory and tissue remodeling cascade of events that could lead to endometrosis. The aim of this study, indeed, is to evaluate and describe the local gene expression of genes involved in acute inflammatory response and fibrosis (COL1A1, COL3A1, TNFA, MMP9, IL6, TGFB1 and TGFBR1), together with others associated to immune modulation (DEFB4B, IDO1 and FOXP3), in uterine specimens from mares of different age.ResultsTwenty-five Standardbred mares were involved in the study with age ranging from 7 to 19 years (mean 10.40 ± 4.42). They were divided by age into two groups: G1 (n = 15, less than 10 years old) and G2 (N = 10, greater than 11 years old). Specimens from the uterus’ right horn-body junction were collected and processed for histology evaluation and RT-qPCR assay.Gene expression of DEFB4B, MMP9 and TNFA was higher in younger mares, suggesting a balance in immune modulation and tissue remodeling. Interleukin-6 and COL3A1 gene expressions were greater in older animals, probably indicating inflammatory pathways activation and fibrosis increase. Although no differences in fibrosis and inflammation distribution could be found with histological examination among G1 and G2, our results suggest a possible involvement of DEF4BB in regulating the local immune response in younger mare’s uterus (G1); age may contribute to the dis-regulation of DEFB4B transcription and, indirectly, influence the extracellular matrix homeostasis. Transcription of IDO1 and FOXP3 genes, instead, does not seem to be age related, or to be involved in local immune-response and tissue remodeling functions.ConclusionsFurther investigations are needed in order to clarify the interactions between the expression of DEFB4B, IL6, TNFA, COL3A1 and MMP9 and other local signals of immune-modulation and tissue remodeling, in mares in a prospective study design.

Selenomethionine exposure affects chondrogenic differentiation and bone formation in Japanese medaka (Oryzias latipes)

Excess selenium entering the aquatic environment from anthropogenic activities has been associated with developmental abnormalities in fish including skeletal deformities of the head and spine. However, mechanisms of this developmental toxicity have not been well-characterized. In this study, Japanese medaka (Oryzias latipes) embryos were exposed to seleno-l-methionine (Se-Met) in a range of concentrations. Gene expression was evaluated for sex-determining region Y (SRY)-related box (Sox9a and Sox9b), runt-related transcription factor 2 (Runx2), and melatonin receptor (Mtr). Alterations in the length of Meckel’s cartilage, tail curvature, and decreased calcification were observed in skeletal stains at 10- and 22-days post-fertilization (dpf). Embryonic exposure of Osterix-mCherry transgenic medaka resulted in fewer teeth. Sox9a and Sox9b were up-regulated, while Runx2 and Mtr were down-regulated by Se-Met prior to hatch. Whole mount in situ hybridization (WISH) localized gene expression to areas observed to be affected in vivo. In addition, Se-Met exposures of a Mtr morpholino (Mtr-MO) as well as Luzindole exposed embryos developed similar skeletal malformations, supporting involvement of Mtr. These findings demonstrate that Se-Met modulates expression of key genes involved in chondrogenic differentiation and bone formation during development.

The Inherited Platelet Disorder of RUNX1 Haploinsufficiency (Familial Platelet Disorder with Associated Myeloid Malignancy (FPDMM)) Has a Marked Deficiency of Megakaryocyte-Biased Hematopoietic Progenitor Cells: Mechanistic Studies and Drug Correction

FPDMM is an autosomal dominant disorder associated with qualitative and quantitative platelet defects and an elevated risk of developing acute myeloid leukemia (AML). FPDMM is due to haploinsufficiency of RUNX1, a transcription factor critical for definitive hematopoiesis and megakaryopoiesis. Currently, there are no drugs available for preventing either the bleeding diathesis or AML predisposition in FPDMM. We hypothesize that drugs that correct RUNX1 expression and/or activity in megakaryopoiesis would not only correct the platelet defect, but prevent leukemia progression. In our studies, we established two cell lines with isogenic controls from induced pluripotent stem cells (iPSC) to study the effects of RUNX1 haploinsufficiency in hematopoietic progenitor cells (iHPCs) and megakaryocytes (iMKs). The first iPSC line (FPD) was derived from patient cells harboring a splice mutation in RUNX1 and its isogenic control was created using the CRISPR/Cas9 technology (FPD-cor). The second line was a normal control iPSC line (WT6) in which the FPD splicing mutation was created by CRISPR/Cas9 gene editing (WT6-mut). When differentiated to iMKs, both lines recapitulated a disease phenotype showing decreased iMK yield per iHPC compared to their respective isogenic controls (29% and 42%, respectively). This decreased yield appeared to be due to depletion of a novel CD42+-iHPC subpopulation in both mutant lines compared to their isogenic controls (5% of isogenic controls (Fig. 1A)). Not shown, these HPCs are von Willibrand factor-positive and almost exclusively gives rise to Mks, thus we termed them Mk-biased. To investigate global and local gene expression changes underlying this depletion, single cell RNA sequencing was performed on sorted CD42+-iHPCs derived from the FPD and FPD-cor lines. In both, we defined 7 identical separate subpopulations, several of which were markedly deficient in the FPD CD42+-HPCs (Fig. 1B). Gene set enrichment analysis (GSEA) of genes upregulated in FPD CD42+-iHPCs from the Gene Ontology and Reactome databases indicates that regulation of response to stress and positive regulation of gene expression gene sets are enriched in FPD CD42+-iHPCs (Fig. 1C, shaded in red). Previously, RepSox, a TGFβ receptor 1 inhibitor, was described by others to enhance megakaryopoiesis in humans and mice, and to increase RUNX1 levels. We now show that RepSox can correct the deficient Mk-biased-HPC population (Fig. 1A) as well as return final iMK yield (not shown) to near FPD-cor levels. GSEA of genes downregulated in RepSox-treated FPD CD42+-iHPCs revealed enrichment of stress-response genes(Fig. 1C, shaded in blue). The stress-activated protein kinase c-Jun N-terminal kinase (JNK) is known to be activated by TGFβ1, various cellular stressors, and pro-inflammatory cytokines. Immunoblotting studies of FPD iMKs showed an elevation in baseline JNK2 phosphorylation (p-JNK) levels (Fig. 1D, left), which was not due to alteration in total JNK2 protein compared to isogenic control iMKs. Immunofluorescence microscopy studies supported these findings (Fig. 1D, right). In summary, we describe a novel defect in the inherited platelet disorder FPDMM by studying iPSC-derived hematopoietic cells. We note a marked deficiency of an MK-biased HPC population that may be related to a proinflammatory state in these HPCs potentially involving TGFβ1-related pathways. These findings need to be confirmed in other FPDMM patient iPSCs and show that it also applies to adult hematopoiesis. Whether the described defect is solely related to the observed thrombocytopenia or also is related to the observed AML defect needs also to be addressed. Disclosures No relevant conflicts of interest to declare.

The Firre locus produces a trans-acting RNA molecule that functions in hematopoiesis

RNA has been classically known to play central roles in biology, including maintaining telomeres, protein synthesis, and in sex chromosome compensation. While thousands of long noncoding RNAs (lncRNAs) have been identified, attributing RNA-based roles to lncRNA loci requires assessing whether phenotype(s) could be due to DNA regulatory elements, transcription, or the lncRNA. Here, we use the conserved X chromosome lncRNA locus Firre, as a model to discriminate between DNA- and RNA-mediated effects in vivo. We demonstrate that (i) Firre mutant mice have cell-specific hematopoietic phenotypes, and (ii) upon exposure to lipopolysaccharide, mice overexpressing Firre exhibit increased levels of pro-inflammatory cytokines and impaired survival. (iii) Deletion of Firre does not result in changes in local gene expression, but rather in changes on autosomes that can be rescued by expression of transgenic Firre RNA. Together, our results provide genetic evidence that the Firre locus produces a trans-acting lncRNA that has physiological roles in hematopoiesis.

DNA methylation analysis of negative pressure therapy effect in diabetic foot ulcers.

ObjectiveNegative pressure wound therapy (NPWT) has been used to treat diabetic foot ulcerations (DFUs). Its action on the molecular level, however, is only partially understood. Some earlier data suggested NPWT may be mediated through modification of local gene expression. As methylation is a key epigenetic regulatory mechanism of gene expression, we assessed the effect of NPWT on its profile in patients with type 2 diabetes (T2DM) and neuropathic non-infected DFUs.MethodsOf 36 included patients, 23 were assigned to NPWT and 13 to standard therapy. Due to ethical concerns, the assignment was non-randomized and based on wound characteristics. Tissue samples were obtained before and 8 ± 1 days after therapy initiation. DNA methylation patterns were checked by Illumina Methylation EPIC kit.ResultsIn terms of clinical characteristics, the groups presented typical features of T2DM; however, the NPWT group had significantly greater wound area: 16.8 cm2 vs 1.4 cm2 (P = 0.0003). Initially only one region at chromosome 5 was differentially methylated. After treatment, 57 differentially methylated genes were found, mainly located on chromosomes 6 (chr6p21) and 20 (chr20p13); they were associated with DNA repair and autocrine signaling via retinoic acid receptor. We performed differential analyses pre treatment and post treatment. The analysis revealed 426 differentially methylated regions in the NPWT group, but none in the control group. The enrichment analysis showed 11 processes significantly associated with NPWT, of which 4 were linked with complement system activation. All but one were hypermethylated after NPWT.ConclusionThe NPWT effect on DFUs may be mediated through epigenetic changes resulting in the inhibition of complement system activation.

Human induced pluripotent stem cell line with genetically encoded fluorescent voltage indicator generated via CRISPR for action potential assessment post-cardiogenesis.

Genetically encoded fluorescent voltage indicators, such as ArcLight, have been used to report action potentials (APs) in human induced pluripotent stem cell-derived cardiomyocytes (hiPSC-CMs). However, the ArcLight expression, in all cases, relied on a high number of lentiviral vector-mediated random genome integrations (8-12 copy/cell), raising concerns such as gene disruption and alteration of global and local gene expression, as well as loss or silencing of reporter genes after differentiation. Here, we report the use of clustered regularly interspaced short palindromic repeats (CRISPR)/Cas9 nuclease technique to develop a hiPSC line stably expressing ArcLight from the AAVS1 safe harbor locus. The hiPSC line retained proliferative ability with a growth rate similar to its parental strain. Optical recording with conventional epifluorescence microscopy allowed the detection of APs as early as 21 days postdifferentiation, and could be repeatedly monitored for at least 5 months. Moreover, quantification and analysis of the APs of ArcLight-CMs identified two distinctive subtypes: a group with high frequency of spontaneous APs of small amplitudes that were pacemaker-like CMs and a group with low frequency of automaticity and large amplitudes that resembled the working CMs. Compared with FluoVolt voltage-sensitive dye, although dimmer, the ArcLight reporter exhibited better optical performance in terms of phototoxicity and photostability with comparable sensitivities and signal-to-noise ratios. The hiPSC line with targeted ArcLight engineering design represents a useful tool for studying cardiac development or hiPSC-derived cardiac disease models and drug testing.

Accurate genome-wide predictions of spatio-temporal gene expression during embryonic development.

Comprehensive information on the timing and location of gene expression is fundamental to our understanding of embryonic development and tissue formation. While high-throughput in situ hybridization projects provide invaluable information about developmental gene expression patterns for model organisms like Drosophila, the output of these experiments is primarily qualitative, and a high proportion of protein coding genes and most non-coding genes lack any annotation. Accurate data-centric predictions of spatio-temporal gene expression will therefore complement current in situ hybridization efforts. Here, we applied a machine learning approach by training models on all public gene expression and chromatin data, even from whole-organism experiments, to provide genome-wide, quantitative spatio-temporal predictions for all genes. We developed structured in silico nano-dissection, a computational approach that predicts gene expression in >200 tissue-developmental stages. The algorithm integrates expression signals from a compendium of 6,378 genome-wide expression and chromatin profiling experiments in a cell lineage-aware fashion. We systematically evaluated our performance via cross-validation and experimentally confirmed 22 new predictions for four different embryonic tissues. The model also predicts complex, multi-tissue expression and developmental regulation with high accuracy. We further show the potential of applying these genome-wide predictions to extract tissue specificity signals from non-tissue-dissected experiments, and to prioritize tissues and stages for disease modeling. This resource, together with the exploratory tools are freely available at our webserver http://find.princeton.edu, which provides a valuable tool for a range of applications, from predicting spatio-temporal expression patterns to recognizing tissue signatures from differential gene expression profiles.

Immune cell infiltrate-associated dysregulation of DNA repair machinery may predispose to papillary thyroid carcinogenesis

BackgroundAn altered immune microenvironment may contribute to papillary thyroid cancer development, as immune infiltrates are identified postoperatively in many papillary thyroid cancer cases with or without diagnosed thyroiditis. Oxygen radicals, endogenous or inflammation-induced, can generate DNA damage, which causes mutations when repaired incorrectly. We hypothesized that infiltrating immune cells might promote aberrant DNA repair, predisposing thyrocytes to papillary thyroid cancer. MethodsQuantitative reverse-transcriptase polymerase chain reaction assays measured gene expression in fresh-frozen samples (n = 55). RNA-seq data was obtained for papillary thyroid cancer and normal thyroid samples from the Cancer Genome Atlas (n = 564), and Hashimoto’s-affected and normal thyroids from the Genotype-Tissue Expression project (n = 279). Immune cell marker expression levels were compared to histological estimates and to selected DNA repair genes. Immunohistochemistry localized gene expression to specific cell types. ResultsDNA polymerase theta expression by quantitative reverse-transcriptase Polymerase chain reaction was higher in papillary thyroid cancer and papillary thyroid cancer-adjacent samples than in benign normal thyroid (P < .001). Immune markers including CD4 correlated with DNA polymerase theta expression (r = 0.50) but not other DNA repair genes examined. Benign tissue with Hashimoto’s exhibited increased DNA polymerase theta (P < .0001) and CD3E (P < .0001) expression. DNA polymerase theta localized to thyrocytes, not lymphocytes. ConclusionWe identified a strong correlation between immune cell infiltrate and dysregulated thyrocyte DNA repair genes, likely reflecting a pathway to papillary thyroid cancer development.

Functional genetic variants can mediate their regulatory effects through alteration of transcription factor binding

Functional variants in the genome are usually identified by their association with local gene expression, DNA methylation or chromatin states. DNA sequence motif analysis and chromatin immunoprecipitation studies have provided indirect support for the hypothesis that functional variants alter transcription factor binding to exert their effects. In this study, we provide direct evidence that functional variants can alter transcription factor binding. We identify a multifunctional variant within the TBC1D4 gene encoding a canonical NFκB binding site, and edited it using CRISPR-Cas9 to remove this site. We show that this editing reduces TBC1D4 expression, local chromatin accessibility and binding of the p65 component of NFκB. We then used CRISPR without genomic editing to guide p65 back to the edited locus, demonstrating that this re-targeting, occurring ~182 kb from the gene promoter, is enough to restore the function of the locus, supporting the central role of transcription factors mediating the effects of functional variants.

Novel Candidates for Vaccine Development Against Mycoplasma Capricolum Subspecies Capripneumoniae (Mccp)-Current Knowledge and Future Prospects.

Exploration of novel candidates for vaccine development against Mycoplasma capricolum subspecies capripneumoniae (Mccp), the causative agent of contagious caprine pleuropneumonia (CCPP), has recently gained immense importance due to both the increased number of outbreaks and the alarming risk of transboundary spread of disease. Treatment by antibiotics as the only therapeutic strategy is not a viable option due to pathogen persistence, economic issues, and concerns of antibiotic resistance. Therefore, prophylactics or vaccines are becoming important under the current scenario. For quite some time inactivated, killed, or attenuated vaccines proved to be beneficial and provided good immunity up to a year. However, their adverse effects and requirement for larger doses led to the need for production of large quantities of Mccp. This is challenging because the required culture medium is costly and Mycoplasma growth is fastidious and slow. Furthermore, quality control is always an issue with such vaccines. Currently, novel candidate antigens including capsular polysaccharides (CPS), proteins, enzymes, and genes are being evaluated for potential use as vaccines. These have shown potential immunogenicity with promising results in eliciting protective immune responses. Being easy to produce, specific, effective and free from side effects, these novel vaccine candidates can revolutionize vaccination against CCPP. Use of novel proteomic approaches, including sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS-PAGE), two-dimensional gel electrophoresis, immunoblotting, matrix-assisted laser desorption/ionization-time-of-flight (MALDI-TOF) mass spectrometry, tandem mass spectroscopy, fast protein liquid chromatography (FPLC), bioinformatics, computerized simulation and genomic approaches, including multilocus sequence analysis, next-generation sequencing, basic local alignment search tool (BLAST), gene expression, and recombinant expression, will further enable recognition of ideal antigenic proteins and virulence genes with vaccination potential.

Localized Osteoarthritis Disease-Modifying Changes due to Intra-articular Injection of Micronized Dehydrated Human Amnion/Chorion Membrane

Osteoarthritis (OA) is the leading cause of joint disability, and there are no FDA-approved disease-modifying drugs. The intra-articular delivery of micronized dehydrated human amnion/chorion membrane (AmnioFix, MiMedx, GA) has been shown to have a chondro-protective effect on articular cartilage in the medial meniscus transection (MMT) pre-clinical model of OA and has entered human clinical trials. AmnioFix is a well-characterized extracellular matrix (ECM)-derived therapeutic that contains hundreds of bioactive molecules, but little is known about its therapeutic mechanism in OA. The objective of this study was to elucidate, via local gene expression analysis, the molecular mechanisms of action of AmnioFix during OA development and progression. Lewis rats underwent MMT surgery, and AmnioFix or saline was injected intra-articularly 24 h post-surgery. At 5, 7, and 21 days post-surgery, articular cartilage, synovial membrane, and osteophyte tissues from multiple regions were collected and analyzed by microarray RT-PCR. Results demonstrated regional variation in the effects of amnion treatment on gene expression. Although gene expression was unaltered in articular cartilage and osteophyte tissue, pro- and anti-inflammatory markers were more highly expressed in the medial synovial membrane for the AmnioFix treatment group compared to the saline and naive control groups. These data suggest that the previously observed chondro-protective effects of AmnioFix treatment may be mediated via alteration of the synovial membrane microenvironment. This work is the first to provide insight into the mechanisms of action of AmnioFix within the articular joint space. AmnioFix (MiMedx, Marietta, GA), obtained from placenta, is a promising candidate for treatment of osteoarthritis (OA). AmnioFix has previously demonstrated protection of the cartilage on joint surfaces in an animal model of OA. However, this is the first study to elucidate the mechanisms of action of AmnioFix on multiple tissues within the joint space. Our results demonstrated that AmnioFix acts primarily through the synovial membrane, where it induces the expression of immunomodulatory markers. We suggest that the immunomodulatory properties of AmnioFix influence the microenvironment of the synovial membrane, thereby affecting joint homeostasis and inducing a chondro-protective effect on the articular cartilage. Future work should investigate the phenotype/s of cells recruited to the synovium following AmnioFix treatment, as well as the interaction of AmnioFix with synovial tissue-resident cells. Furthermore, the effects of AmnioFix on other joint disorders such as rheumatoid arthritis, a disease involving inflammation of the synovium, warrants further investigation.

Active Immunoprophylaxis and Vaccine Augmentations Mediated by a Novel Plasmid DNA Formulation.

Plasmid DNA (pDNA) gene delivery is a highly versatile technology that has the potential to address a multitude of unmet medical needs. Advances in pDNA delivery to host tissue with the employment of in vivo electroporation (EP) have led to significantly enhanced gene expression and the recent demonstration of clinical efficacy with the platform. Building upon this platform, this study reports that enzyme-mediated modification of the muscle tissue extracellular matrix structure at the site of pDNA delivery operates in a synergistic manner with EP to enhance both local and systemic gene expression further. Specifically, administration of chondroitinase ABC (Cho ABC) to the site of intramuscular delivery of pDNA led to transient disruption of chondroitin sulfate scaffolding barrier, permitting enhanced gene distribution and expression across the tissue. The employment of Cho ABC in combination with CELLECTRA® intramuscular EP resulted in increased gene expression by 5.5-fold in mice and 17.98-fold in rabbits. The study demonstrates how this protocol can be universally applied to an active prophylaxis platform to increase the in vivo production of functional immunoglobulin G, and to DNA vaccine protocols to permit drug dose sparing. The data indicate the Cho ABC formulation to be of significant value upon combination with EP to drive enhanced gene expression levels in pDNA delivery protocols.

Molecular Mechanisms of Cue‐Induced Translation Regulation

Precise tuning of local gene expression is essential for proper neural development. In particular, environmental cues spatially and temporally direct neuron growth by stimulating or repressing protein synthesis. The integral membrane protein, deleted in colorectal cancer, or DCC, detects the environmental growth cue netrin‐1, and transduces the growth signal to a variety of intracellular responses, including activating protein synthesis. DCC is primed to function in this way; in the absence of netrin‐1, translation machinery is tethered to its C‐terminal tail (C‐tail) and silenced. Once netrin‐1 binds DCC, the receptor dimerizes, translation machinery is released, and protein synthesis commences. How DCC's C‐tail binds to the protein synthesis machinery and prevents translation in the absence of netrin‐1 is unknown. The C‐tail is a 35 kDa protein that is composed of three domains (P1–3) and is predicted 91% unstructured. Dimerization is facilitated by the P3 domain, while the P1 domain binds to the 60S subunit protein, ribosomal protein L5 (rpL5). Titrating the full‐length C‐tail into translation‐competent lysates, we found that the tail specifically inhibits eukaryotic translation, and inhibits protein synthesis of messages that use both canonical and non‐canonical translation initiation mechanisms. Hence, we propose that the C‐tail functions primarily through binding the ribosome, not eukaryotic initiation factors. We have evidence from cosedimentation and in vitro binding studies to show that the full‐length tail binds to the small and large subunits, as well as the 80S ribosome in the absence of mRNA. Binding and luciferase reporter assays show that the P3 dimerization domain is dispensable for translation inhibition, and the P1 domain is necessary but not sufficient to bind the ribosome and decrease translation. Thus the P2 domain and/or linker sequence(s) are also important for function. Collectively, our results suggest that DCC's C‐tail inhibits translation by effectively preventing ribosomes from binding to messages. We hypothesize the unstructured C‐tail precludes mRNA binding by interacting with the decoding groove of the ribosome until receptor dimerization releases 80S ribosomes. Thus chemoattraction via netrin‐induced DCC receptor dimerization might lead to a global (not message‐specific) increase in localized protein synthesis at axonal growth cones.Support or Funding InformationMSU Denver Provost Seed GrantMSU Denver College of Letters, Arts and Sciences Dean's Seed GrantThis abstract is from the Experimental Biology 2019 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.

DNA Methylation Patterns of Interferon Gamma Gene Promoter and Serum Level in Pulmonary Tuberculosis: Their Role in Prognosis

Tuberculosis (TB) still remains an important medical problem due to high levels of morbidity and mortality worldwide. A series of innate immune mechanisms that create a cytokine network control the pathogenesis of tuberculosis and this response has the capacity to modify the host genomic DNA structure through epigenetic mechanisms such as DNA methylation which could constantly alter the local gene expression pattern that can modulate the metabolism of the tissues and the immune-response. Interferon-gamma (IFN-γ) is an important pro-inflammatory cytokine regulator of the innate immune response to TB. This study aims to determine DNA methylation patterns of INF-γ gene promoter and measure serum IFN- γ level in newly diagnosed TB patients, relapse TB patients, and healthy control, in order to study the possibility of using these as a biomarker for the prognosis of TB stages in patients. The current case-control study included 66 patients with TB and 33 healthy control subjects. DNA was extracted from peripheral blood(PB) of included subjects and modified using sodium bisulfate specific kit. DNA methylation patterns of IFN-γ gene promoter was determine by using methylation specific polymerase chain reaction(MS-PCR).Serum IFN-γ level was determined using enzyme linked immune-sorbent assay(ELISA). Results showed that percentages of DNA methylation patterns in normal controls, newly diagnostic TB patients and relapse TB patients were (63.3%, 18.2% and 21.2% respectively). Also, higher significant differences (P≤0.0001) of un-methylated IFN-γ gene promoter patterns in newly diagnostic TB patients than relapse TB patients comparison with healthy controls. The percentage of un-methylated DNA patterns in healthy controls, newly diagnostic TB patients and relapse TB patients were (9.9%, 39.4% and 51.5%, respectively). The mean of serum IFN-γ levels (pg/ml) for normal controls, newly diagnostic TB patients and relapse TB patients were (59.3± 13.8,75.8±24.3 and 69.6±18.7,respectively).In conclusion, there is a relative association between methylation of IFN-γ gene promoter and predisposing to TB progression.

DNA Methylation Patterns of Interferon Gamma Gene Promoter and Serum Level in Pulmonary Tuberculosis: Their Role in Prognosis

Tuberculosis (TB) still remains an important medical problem due to high levels of morbidity and mortality worldwide. A series of innate immune mechanisms that create a cytokine network control the pathogenesis of tuberculosis and this response has the capacity to modify the host genomic DNA structure through epigenetic mechanisms such as DNA methylation which could constantly alter the local gene expression pattern that can modulate the metabolism of the tissues and the immune-response. Interferon-gamma (IFN-γ) is an important pro-inflammatory cytokine regulator of the innate immune response to TB. This study aims to determine DNA methylation patterns of INF-γ gene promoter and measure serum IFN- γ level in newly diagnosed TB patients, relapse TB patients, and healthy control, in order to study the possibility of using these as a biomarker for the prognosis of TB stages in patients. The current case-control study included 66 patients with TB and 33 healthy control subjects. DNA was extracted from peripheral blood(PB) of included subjects and modified using sodium bisulfate specific kit. DNA methylation patterns of IFN-γ gene promoter was determine by using methylation specific polymerase chain reaction(MS-PCR).Serum IFN-γ level was determined using enzyme linked immune-sorbent assay(ELISA). Results showed that percentages of DNA methylation patterns in normal controls, newly diagnostic TB patients and relapse TB patients were (63.3%, 18.2% and 21.2% respectively). Also, higher significant differences (P≤0.0001) of un-methylated IFN-γ gene promoter patterns in newly diagnostic TB patients than relapse TB patients comparison with healthy controls. The percentage of un-methylated DNA patterns in healthy controls, newly diagnostic TB patients and relapse TB patients were (9.9%, 39.4% and 51.5%, respectively). The mean of serum IFN-γ levels (pg/ml) for normal controls, newly diagnostic TB patients and relapse TB patients were (59.3± 13.8,75.8±24.3 and 69.6±18.7,respectively).In conclusion, there is a relative association between methylation of IFN-γ gene promoter and predisposing to TB progression.

Reevaluation of the Role of Extracellular Signal-Regulated Kinase 3 in Perinatal Survival and Postnatal Growth Using New Genetically Engineered Mouse Models.

The physiological functions of the atypical mitogen-activated protein kinase extracellular signal-regulated kinase 3 (ERK3) remain poorly characterized. Previous analysis of mice with a targeted insertion of the lacZ reporter in the Mapk6 locus (Mapk6lacZ ) showed that inactivation of ERK3 in Mapk6lacZ mice leads to perinatal lethality associated with intrauterine growth restriction, defective lung maturation, and neuromuscular anomalies. To further explore the role of ERK3 in physiology and disease, we generated novel mouse models expressing a catalytically inactive (Mapk6KD ) or conditional (Mapk6Δ ) allele of ERK3. Surprisingly, we found that mice devoid of ERK3 kinase activity or expression survive the perinatal period without any observable lung or neuromuscular phenotype. ERK3 mutant mice reached adulthood, were fertile, and showed no apparent health problem. However, analysis of growth curves revealed that ERK3 kinase activity is necessary for optimal postnatal growth. To gain insight into the genetic basis underlying the discrepancy in phenotypes of different Mapk6 mutant mouse models, we analyzed the regulation of genes flanking the Mapk6 locus by quantitative PCR. We found that the expression of several Mapk6 neighboring genes is deregulated in Mapk6lacZ mice but not in Mapk6KD or Mapk6Δ mutant mice. Our genetic analysis suggests that off-target effects of the targeting construct on local gene expression are responsible for the perinatal lethality phenotype of Mapk6lacZ mutant mice.