Cell-specific Patterns Research Articles

The expression and localization of Toll-like receptors 2, 4, 5 and 9 in the epididymis and vas deferens of a adult tom cats

Toll-like receptors (TLRs) are important molecules, which provide protection against infections of the reproductive tract. This study demonstrates for the first time the expression and localization patterns of TLRs in the caput, corpus and cauda segments of the epididymal duct (ED) and the vas deferens (VD) of adult domestic cats using immunohistochemistry and western blotting. While immunoblot analyses revealed relatively similar protein levels for TLRs 2, 4, 5, and 9 in three segments of the ED, the protein levels of TLR2 and TLR4 in the VD were found to be significantly higher than those measured in the ED segments (P < 0.05). On the other hand, immunostaining showed that TLRs exhibited regional- and cell-specific localization patterns. TLR2 and TLR5 were immunolocalized to the nucleus and cytoplasm of the principal cells in all ducts. TLR4 was restricted to the stereocilia, and TLR9 was located in the cytoplasm of the principal cells. Narrow cells displayed positive immunoreactions for TLR4 and TLR5. The basal cells of the different ED segments were positive for all four TLRs. TLR2, TLR5 and TLR9 were detected in the cytoplasmic droplets of the spermatozoa. TLR4 and TLR9 were detected along the entire length of the sperm tail, whilst TLR2 and TLR5 were absent in the midpiece. TLR2 and TLR5 were also detected in the equatorial segment of the sperm head. These results suggest that TLR2, TLR4, TLR5 and TLR9 are important not only for the protection of the ED, VD and spermatozoa but also for the maturation and storage of spermatozoa in the ED and VD, respectively.

Single-cell transcriptomics from human pancreatic islets: sample preparation matters.

Single-cell RNA sequencing (scRNA-seq) of human primary tissues is a rapidly emerging tool for investigating human health and disease at the molecular level. However, optimal processing of solid tissues presents a number of technical and logistical challenges, especially for tissues that are only available at autopsy, which includes pancreatic islets, a tissue that is highly relevant to diabetes. To assess the possible effects of different sample preparation protocols on fresh islet samples, we performed a detailed comparison of scRNA-seq data generated with islets isolated from a human donor but processed according to four treatment strategies, including fixation and cryopreservation. We found significant and reproducible differences in the proportion of cell types identified, and more minor effects on cell-specific patterns of gene expression. Fresh islets from a second donor confirmed gene expression signatures of alpha and beta subclusters. These findings may well apply to other tissues, emphasizing the need for careful consideration when choosing processing methods, comparing results between different studies, and/or interpreting data in the context of multiple cell types from preserved tissue.

Evolution of host support for two ancient bacterial symbionts with differentially degraded genomes in a leafhopper host

Plant sap-feeding insects (Hemiptera) rely on bacterial symbionts for nutrition absent in their diets. These bacteria experience extreme genome reduction and require genetic resources from their hosts, particularly for basic cellular processes other than nutrition synthesis. The host-derived mechanisms that complete these processes have remained poorly understood. It is also unclear how hosts meet the distinct needs of multiple bacterial partners with differentially degraded genomes. To address these questions, we investigated the cell-specific gene-expression patterns in the symbiotic organs of the aster leafhopper (ALF), Macrosteles quadrilineatus (Cicadellidae). ALF harbors two intracellular symbionts that have two of the smallest known bacterial genomes: Nasuia (112 kb) and Sulcia (190 kb). Symbionts are segregated into distinct host cell types (bacteriocytes) and vary widely in their basic cellular capabilities. ALF differentially expresses thousands of genes between the bacteriocyte types to meet the functional needs of each symbiont, including the provisioning of metabolites and support of cellular processes. For example, the host highly expresses genes in the bacteriocytes that likely complement gene losses in nucleic acid synthesis, DNA repair mechanisms, transcription, and translation. Such genes are required to function in the bacterial cytosol. Many host genes comprising these support mechanisms are derived from the evolution of novel functional traits via horizontally transferred genes, reassigned mitochondrial support genes, and gene duplications with bacteriocyte-specific expression. Comparison across other hemipteran lineages reveals that hosts generally support the incomplete symbiont cellular processes, but the origins of these support mechanisms are generally specific to the host-symbiont system.

Integrative Analysis of Three RNA Sequencing Methods Identifies Mutually Exclusive Exons of MADS-Box Isoforms During Early Bud Development in Picea abies.

Recent efforts to sequence the genomes and transcriptomes of several gymnosperm species have revealed an increased complexity in certain gene families in gymnosperms as compared to angiosperms. One example of this is the gymnosperm sister clade to angiosperm TM3-like MADS-box genes, which at least in the conifer lineage has expanded in number of genes. We have previously identified a member of this sub-clade, the conifer gene DEFICIENS AGAMOUS LIKE 19 (DAL19), as being specifically upregulated in cone-setting shoots. Here, we show through Sanger sequencing of mRNA-derived cDNA and mapping to assembled conifer genomic sequences that DAL19 produces six mature mRNA splice variants in Picea abies. These splice variants use alternate first and last exons, while their four central exons constitute a core region present in all six transcripts. Thus, they are likely to be transcript isoforms. Quantitative Real-Time PCR revealed that two mutually exclusive first DAL19 exons are differentially expressed across meristems that will form either male or female cones, or vegetative shoots. Furthermore, mRNA in situ hybridization revealed that two mutually exclusive last DAL19 exons were expressed in a cell-specific pattern within bud meristems. Based on these findings in DAL19, we developed a sensitive approach to transcript isoform assembly from short-read sequencing of mRNA. We applied this method to 42 putative MADS-box core regions in P. abies, from which we assembled 1084 putative transcripts. We manually curated these transcripts to arrive at 933 assembled transcript isoforms of 38 putative MADS-box genes. 152 of these isoforms, which we assign to 28 putative MADS-box genes, were differentially expressed across eight female, male, and vegetative buds. We further provide evidence of the expression of 16 out of the 38 putative MADS-box genes by mapping PacBio Iso-Seq circular consensus reads derived from pooled sample sequencing to assembled transcripts. In summary, our analyses reveal the use of mutually exclusive exons of MADS-box gene isoforms during early bud development in P. abies, and we find that the large number of identified MADS-box transcripts in P. abies results not only from expansion of the gene family through gene duplication events but also from the generation of numerous splice variants.

Evolution of Chikungunya virus in mosquito cells

It has been observed that replication of Chikungunya virus (CHIKV) in C6/36 Aedes albopictus cells has little effect on virus evolution. To characterize evolutionary patterns associated with CHIKV replication in mosquito cells, we performed serial passages of the LR2006 strain in Ae. albopictus cells (75 and 30 passages in C6/36 and U4.4 respectively) and Ae. aegypti cells (100 passages in AA-A20 and in AE) and studied genotypic changes accompanying adaptation during this evolutionary process. Quantitative analysis revealed cell specific patterns: low mutation rates in C6/36 cells except when a CHIKV strain pre-adapted to mammalian was used and typical features of adaptation to cell culture conditions with a high number of fixed mutations in AE and AA-A20 cells probably due to the weak permissiveness of these latter cell lines. Altogether, these results suggested that both cell line and viral strain influence rates of viral evolution. In contrast, characteristics and distribution of mutations were qualitatively very similar in all mosquito cells with a high level of parallel evolution including 4 deletion mutations. Serial passage in mammalian cells of viruses pre-adapted to mosquito cells revealed disappearance of almost all shared mutations suggesting that many of these mutational patterns are vector-specific.

Single-cell transcriptome profiling of the Ciona larval brain

The tadpole-type larva of Ciona has emerged as an intriguing model system for the study of neurodevelopment. The Ciona intestinalis connectome has been recently mapped, revealing the smallest central nervous system (CNS) known in any chordate, with only 177 neurons. This minimal CNS is highly reminiscent of larger CNS of vertebrates, sharing many conserved developmental processes, anatomical compartments, neuron subtypes, and even specific neural circuits. Thus, the Ciona tadpole offers a unique opportunity to understand the development and wiring of a chordate CNS at single-cell resolution. Here we report the use of single-cell RNAseq to profile the transcriptomes of single cells isolated by fluorescence-activated cell sorting (FACS) from the whole brain of Ciona robusta (formerly intestinalis Type A) larvae. We have also compared these profiles to bulk RNAseq data from specific subsets of brain cells isolated by FACS using cell type-specific reporter plasmid expression. Taken together, these datasets have begun to reveal the compartment- and cell-specific gene expression patterns that define the organization of the Ciona larval brain.

The State of Long Non-Coding RNA Biology

Transcriptomic studies have demonstrated that the vast majority of the genomes of mammals and other complex organisms is expressed in highly dynamic and cell-specific patterns to produce large numbers of intergenic, antisense and intronic long non-protein-coding RNAs (lncRNAs). Despite well characterized examples, their scaling with developmental complexity, and many demonstrations of their association with cellular processes, development and diseases, lncRNAs are still to be widely accepted as major players in gene regulation. This may reflect an underappreciation of the extent and precision of the epigenetic control of differentiation and development, where lncRNAs appear to have a central role, likely as organizational and guide molecules: most lncRNAs are nuclear-localized and chromatin-associated, with some involved in the formation of specialized subcellular domains. I suggest that a reassessment of the conceptual framework of genetic information and gene expression in the 4-dimensional ontogeny of spatially organized multicellular organisms is required. Together with this and further studies on their biology, the key challenges now are to determine the structure–function relationships of lncRNAs, which may be aided by emerging evidence of their modular structure, the role of RNA editing and modification in enabling epigenetic plasticity, and the role of RNA signaling in transgenerational inheritance of experience.

Dynamic expression patterns of Irx3 and Irx5 during germline nest breakdown and primordial follicle formation promote follicle survival in mouse ovaries.

Women and other mammalian females are born with a finite supply of oocytes that determine their reproductive lifespan. During fetal development, individual oocytes are enclosed by a protective layer of granulosa cells to form primordial follicles that will grow, mature, and eventually release the oocyte for potential fertilization. Despite the knowledge that follicles are dysfunctional and will die without granulosa cell-oocyte interactions, the mechanisms by which these cells establish communication is unknown. We previously identified that two members of the Iroquois homeobox transcription factor gene family, Irx3 and Irx5, are expressed within developing ovaries but not testes. Deletion of both factors (Irx3-Irx5EGFP/Irx3-Irx5EGFP) disrupted granulosa cell-oocyte contact during early follicle development leading to oocyte death. Thus, we hypothesized that Irx3 and Irx5 are required to develop cell-cell communication networks to maintain follicle integrity and female fertility. A series of Irx3 and Irx5 mutant mouse models were generated to assess roles for each factor. While both Irx3 and Irx5 single mutant females were subfertile, their breeding outcomes and ovary histology indicated distinct causes. Careful analysis of Irx3- and Irx5-reporter mice linked the cause of this disparity to dynamic spatio-temporal changes in their expression patterns. Both factors marked the progenitor pre-granulosa cell population in fetal ovaries. At the critical phase of germline nest breakdown and primordial follicle formation however, Irx3 and Irx5 transitioned to oocyte- and granulosa cell-specific expression respectively. Further investigation into the cause of follicle death in Irx3-Irx5EGFP/Irx3-Irx5EGFP ovaries uncovered specific defects in both granulosa cells and oocytes. Granulosa cell defects included poor contributions to basement membrane deposition and mis-localization of gap junction proteins. Granulosa cells and oocytes both presented fewer cell projections resulting in compromised cell-cell communication. Altogether, we conclude that Irx3 and Irx5 first work together to define the pregranulosa cell population of germline nests. During primordial follicle formation, they transition to oocyte- and granulosa cell-specific expression patterns where they cooperate in neighboring cells to build the foundation for follicle integrity. This foundation is left as their legacy of the essential oocyte-granulosa cell communication network that ensures and ultimately optimizes the integrity of the ovarian reserve and therefore, the female reproductive lifespan.

Cell-specific pattern of berberine pleiotropic effects on different human cell lines

The natural alkaloid berberine has several pharmacological properties and recently received attention as a potential anticancer agent. In this work, we investigated the molecular mechanisms underlying the anti-tumor effect of berberine on glioblastoma U343 and pancreatic carcinoma MIA PaCa-2 cells. Human dermal fibroblasts (HDF) were used as non-cancer cells. We show that berberine differentially affects cell viability, displaying a higher cytotoxicity on the two cancer cell lines than on HDF. Berberine also affects cell cycle progression, senescence, caspase-3 activity, autophagy and migration in a cell-specific manner. In particular, in HDF it induces cell cycle arrest in G2 and senescence, but not autophagy; in the U343 cells, berberine leads to cell cycle arrest in G2 and induces both senescence and autophagy; in MIA PaCa-2 cells, the alkaloid induces arrest in G1, senescence, autophagy, it increases caspase-3 activity and impairs migration/invasion. As demonstrated by decreased citrate synthase activity, the three cell lines show mitochondrial dysfunction following berberine exposure. Finally, we observed that berberine modulates the expression profile of genes involved in different pathways of tumorigenesis in a cell line-specific manner. These findings have valuable implications for understanding the complex functional interactions between berberine and specific cell types.

Connexin 30.2 is expressed in exocrine vascular endothelial and ductal epithelial cells throughout pancreatic postnatal development

Previously we have demonstrated that the GJ protein connexin 30.2 (Cx30.2) is expressed in pancreatic beta cells and endothelial cells (ECs) of the islet. In the present study, we address whether Cx30.2 is expressed in the exocrine pancreas, including its vascular system. For this, adult mouse pancreatic sections were double labeled with specific antibodies against Cx30.2 and CD31, an endothelial cell marker, or with anti-α-actin smooth muscle, a smooth muscle cell (SMC) marker or anti-mucin-1, a marker of epithelial ductal cells, using immunofluorescence (IF) studies. Cx30.2-IF hot spots were found at junctional membranes of exocrine ECs and SMCs of blood vessels. Furthermore, Cx30.2 was localized in mucin-1 positive cells or epithelial ductal cells. Using immunohistochemistry (IHC) studies, it was found that in vessels and ducts of different diameters, Cx30.2 was also expressed in these cell types. In addition, it was found that Cx30.2 is already expressed in these cell types in pancreatic sections of 3, 14 and 21 days postpartum. Moreover, this cell specific pattern of expression was also found in the adult rat, hamster and guinea pig pancreas. Expression of Cx30.2 mRNA and protein in the pancreas of all these species was confirmed by RT-PCR and Western blot studies. Overall, our results suggest that intercellular coupling mediated by Cx30.2 intercellular channels may synchronize the functional activity of ECs and SMCs of vascular cells, as well as of epithelial ductal cells after birth.

Heterodimeric BMP-2/7 exhibits different osteoinductive effects in human and murine cells

As robust osteoinductive cytokines, bone morphogenetic proteins (BMPs) play a significant role in bone tissue engineering. Constituted of two different polypeptides, heterodimeric BMPs are more effective than the homodimers in bone formation. While most studies focused on the murine cell lines, such as murine preosteoblasts MC3T3-E1, the role of heterodimeric BMPs in the osteogenic differentiation of human cells remains uncertain, which hinders their application to practical treatment. In this study, we compared the osteoinductive effects of BMP-2/7 heterodimer in human adipose-derived stem cells (hASCs) with their homodimers BMP-2 and BMP-7, in which MC3T3-E1 cells were utilized as a positive control. The results indicated that BMP-2/7 was not a stronger inducer during the osteogenic differentiation of hASCs as that for MC3T3-E1, and extracellular-signal-regulated kinase signaling played a role in the different effects of BMP-2/7 between hASCs and MC3T3-E1. Our study demonstrates the osteoinductive effects of heterodimeric BMP-2/7 present in a cell-specific pattern and cautions should be taken when applying heterodimeric BMP-2/7 to clinical practice.

Abstract 4716: Targeting specific TGF-β isoforms in combination with radiation therapy leads to differential antitumor effects in mouse models of cancer

Abstract Background: TGF-β is a pleotropic cytokine, which has emerged as a potential target in cancer treatment due to its dual role in tumorigenesis and homeostasis. There are three isoforms of TGF-β (TGF-β1, TGF-β2 and TGF-β3), which are secreted by immune and nonimmune cells as a latent complex. Depending on the local context, TGF-β adopts opposing roles in carcinogensis and in modulating the immune system. These dueling roles of TGF-β are dependent on its secretion and activation. Local radiation therapy (RT) can activate TGF-β via reactive oxygen species. Such TGF-β expression is linked to radioresistance and dose-limiting toxicities, reducing the effectiveness of RT. In these studies, we aim to characterize the effect of RT on the temporal and cell-specific expression patterns of TGF-β isoforms in mouse tumor models. This will inform treatment regimens combining isoform specific anti-TGF-β therapy with RT. Methods: Fluorescence-activated cell sorting (FACS): C57BL/6 mice were implanted on the hind limb with B16-F10 melanoma cells. On day 10, tumors were irradiated locally with 15 Gy. Expression of TGF-β isoforms was measured at 1, 3 and 5 days post-RT by FACS. In vivo: C57BL/6 mice were implanted with tumors and irradiated as described. Mice were treated (10/group) with anti-TGF-β1, anti-TGF-β3 or a pan-TGF-β antibody beginning 1 day after RT given intraperitoneally (200 ug/mouse) every other day for 8 doses. Tumor growth and overall survival were monitored. A similar experiment was conducted in the 4T1 breast cancer model, in which mice were treated 1 day prior to radiation. Results: FACS data indicated that TGF-β1 and TGF-β3 expression increases on most immune cells in the tumor 1 day after RT, decreases 3 days after RT and reaches a peak 5 days after RT. Preliminary in vivo studies demonstrate that both αTGF-β1 and αTGF-β3 as monotherapies have activity against B16 melanoma. In combination with RT, αTGF-β3 shows greater antitumor activity compared to αTGF-β1 in melanoma. Similar observations were obtained in a 4T1 breast model; however, αTGF-β3 alone and in combination with RT as well as αTGF-β1 + RT showed a significant delay against tumor growth. No significant differences in survival were seen in either tumor model. Conclusions: TGF-β1 and TGF-β3 are expressed on numerous lymphoid and myeloid cells in B16 tumors and spleens. TGF-β isoform expression peaks 5 days post-RT. Anti-TGF-β therapy is effective in delaying tumor growth and may synergize with RT in certain cancers. This demonstrates rationale for the use of anti-TGF-β therapy to enhance the effectiveness of RT in cancer. Citation Format: Aditi Gupta, Sadna Budhu, Rachel Giese, Jacques van Snick, Catherine Uyttenhove, Gerd Ritter, Jedd Wolchok, Taha Merghoub. Targeting specific TGF-β isoforms in combination with radiation therapy leads to differential antitumor effects in mouse models of cancer [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2018; 2018 Apr 14-18; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2018;78(13 Suppl):Abstract nr 4716.

A Systems-Level Analysis Reveals Circadian Regulation of Splicing in Colorectal Cancer

Accumulating evidence points to a significant role of the circadian clock in the regulation of splicing in various organisms, including mammals. Both dysregulated circadian rhythms and aberrant pre-mRNA splicing are frequently implicated in human disease, in particular in cancer. To investigate the role of the circadian clock in the regulation of splicing in a cancer progression context at the systems-level, we conducted a genome-wide analysis and compared the rhythmic transcriptional profiles of colon carcinoma cell lines SW480 and SW620, derived from primary and metastatic sites of the same patient, respectively. We identified spliceosome components and splicing factors with cell-specific circadian expression patterns including SRSF1, HNRNPLL, ESRP1, and RBM 8A, as well as altered alternative splicing events and circadian alternative splicing patterns of output genes (e.g., VEGFA, NCAM1, FGFR2, CD44) in our cellular model. Our data reveals a remarkable interplay between the circadian clock and pre-mRNA splicing with putative consequences in tumor progression and metastasis.

Toll-like receptors 4, 5, 6 and 7 are constitutively expressed in non-human primate retinal neurons

The purpose of this study was to characterize cell-specific expression patterns of Toll-like receptors (TLR) in non-human primate (NHP) neural retina tissue. TLR 4, 5, 6, and 7 proteins were detected by immunblotting of macaque retina tissue lysates and quantitative PCR (qPCR) demonstrated TLRs 4–7 mRNA expression. Immunofluorescence (IF) microscopy detected TLRs 4–7 in multiple cell types in macaque neural retina including Muller, retinal ganglion cells (RGC), amacrine, and bipolar cells. These results demonstrate that TLRs 4–7 are constitutively expressed by neurons in the NHP retina raising the possibility that these cells could be involved in retinal innate inflammatory responses.

MMARGE: Motif Mutation Analysis for Regulatory Genomic Elements.

Cell-specific patterns of gene expression are determined by combinatorial actions of sequence-specific transcription factors at cis-regulatory elements. Studies indicate that relatively simple combinations of lineage-determining transcription factors (LDTFs) play dominant roles in the selection of enhancers that establish cell identities and functions. LDTFs require collaborative interactions with additional transcription factors to mediate enhancer function, but the identities of these factors are often unknown. We have shown that natural genetic variation between individuals has great utility for discovering collaborative transcription factors. Here, we introduce MMARGE (Motif Mutation Analysis of Regulatory Genomic Elements), the first publicly available suite of software tools that integrates genome-wide genetic variation with epigenetic data to identify collaborative transcription factor pairs. MMARGE is optimized to work with chromatin accessibility assays (such as ATAC-seq or DNase I hypersensitivity), as well as transcription factor binding data collected by ChIP-seq. Herein, we provide investigators with rationale for each step in the MMARGE pipeline and key differences for analysis of datasets with different experimental designs. We demonstrate the utility of MMARGE using mouse peritoneal macrophages, liver cells, and human lymphoblastoid cells. MMARGE provides a powerful tool to identify combinations of cell type-specific transcription factors while simultaneously interpreting functional effects of non-coding genetic variation.

P-88 - Involvement of histone acetylation and methylation in SOD3 regulation in human monocytic THP-1 cells

Superoxide dismutase 3 (SOD3), a secretory SOD isozyme, displays a unique and cell-specific expression pattern, and epigenetics plays a critical role in its expression. To date, we have determined that histone acetylation governs phorbol ester (TPA)-elicited SOD3 expression in human monocytic THP-1 cells; however, the molecular mechanisms involving its induction remains unclear. Present data demonstrated that histone deacetylase 1 (HDAC1) and histone acetyltransferase (p300) coordinated SOD3 induction. Moreover, it was determined that myocyte enhancer factor 2 A (MEF2A) and MEF2D function as scaffold proteins, indicating that MEF2/HDAC1 or MEF2/p300 complex regulates SOD3 expression. On the other hand, asymmetric H4R3me2 (H4R3me2a), an epigenetic marker for transcription activation, was also induced in TPA-treated THP-1 cells, suggesting that histone methylation regulates SOD3 expression as well. As expected, our ChIP data clearly showed that the level of H4R3me2a within the proximal SOD3 promoter region was significantly enhanced. Overall, we have found that histone modifications including acetylation and methylation govern SOD3 expression in THP-1 cells.

32.1 MECHANISMS OF ABNORMAL POSTTRANSLATIONAL PROTEIN PROCESSING IN SCHIZOPHRENIA BRAIN

32.1 MECHANISMS OF ABNORMAL POSTTRANSLATIONAL PROTEIN PROCESSING IN SCHIZOPHRENIA BRAIN

Antibody receptors steal the sweet spotlight

Immunoglobulin G (IgG) antibodies function, in part, through ligation of cell-surface Fc receptors such as FcγRIIIA (also known as CD16A). IgG glycosylation is known to impact antibody function, but the role of FcγRIIIA glycans, if any, is unclear. Patel et al. now reveal that these glycans do impact protein conformation and IgG affinity and display cell-specific glycosylation patterns, leading to a potential model in which the affinity and possibly function of Fc receptors is dictated by the cell type and its surface glycome.

Identification of the Regulatory Region Responsible for Vascular Tissue-Specific Expression in the Rice Hd3a Promoter.

Flowering time is determined by florigens. These genes include, Heading date 3a (Hd3a) and Rice FT 1 (RFT1) in rice, which are specifically expressed in the vascular tissues of leaves at the floral transition stage. To study the cis-regulatory elements present in the promoter region of Hd3a, we generated transgenic plants carrying the 1.75-kb promoter fragment of Hd3a that was fused to the β-glucuronidase (GUS) reporter gene. Plants expressing this construct conferred a vascular cell-specific expression pattern for the reporter gene. However, GUS was expressed in leaves at all developmental stages, including the early seedling stage when Hd3a was not detected. Furthermore, the reporter was expressed in roots at all stages. This suggests that the 1.75-kb region lackings cis-elements that regulate leaf-specific expression at the appropriate developmental stages. Deletion analyses of the promoter region indicated that regulatory elements determining vascular cell-specific expression are present in the 200-bp region between -245 bp and -45 bp from the transcription initiation site. By transforming the Hd3a–GUS construct to rice cultivar ‘Taichung 65’ which is defective in Ehd1, we observed that Ehd1 is the major regulatory element that controls Hd3a promoter activity.

Microglial Inflammasome Activation in Penetrating Ballistic-Like Brain Injury.

Penetrating traumatic brain injury (PTBI) is a significant cause of death and disability in the United States. Inflammasomes are one of the key regulators of the interleukin (IL)-1β mediated inflammatory responses after traumatic brain injury. However, the contribution of inflammasome signaling after PTBI has not been determined. In this study, adult male Sprague-Dawley rats were subjected to sham procedures or penetrating ballistic-like brain injury (PBBI) and sacrificed at various time-points. Tissues were assessed by immunoblot analysis for expression of IL-1β, IL-18, and components of the inflammasome: apoptosis-associated speck-like protein containing a caspase-activation and recruitment domain (ASC), caspase-1, X-linked inhibitor of apoptosis protein (XIAP), nucleotide-binding oligomerization domain (NOD)-like receptor protein 3 (NLRP3), and gasdermin-D (GSDMD). Specific cell types expressing inflammasome proteins also were evaluated immunohistochemically and assessed quantitatively. After PBBI, expression of IL-1β, IL-18, caspase-1, ASC, XIAP, and NLRP3 peaked around 48 h. Brain protein lysates from PTBI animals showed pyroptosome formation evidenced by ASC laddering, and also contained increased expression of GSDMD at 48 h after injury. ASC-positive immunoreactive neurons within the perilesional cortex were observed at 24 h. At 48 h, ASC expression was concentrated in morphologically activated cortical microglia. This expression of ASC in activated microglia persisted until 12 weeks following PBBI. This is the first report of inflammasome activation after PBBI. Our results demonstrate cell-specific patterns of inflammasome activation and pyroptosis predominantly in microglia, suggesting a sustained pro-inflammatory state following PBBI, thus offering a therapeutic target for this type of brain injury.