Cloning and functional analysis in transgenic tobacco of a tapetum-specific promoter from Arabidopsis

The 5’-flanking region of 1174 bp upstream of the translation start point (TSP) of a reported Arabidopsis anther-specific gene, Anther7 gene (ATA7), which putatively  encodes a protein related to lipid transfer protein, was cloned and functionally analyzed in transgenic tobacco after been fused with β- glucuronidase (GUS) gene reporter. Histochemical GUS staining of the transgenic plants showed that the cloned fragment did drive GUS expression exclusively in the anther, not  in any other parts of floral organs, including pollens and nor in any vegetative tissue. Transverse section of the GUS-blue anthers disclosed that the blue cells were present uniquely in the tapetum of the anther. A series of 5’-deletion of cloned fragment indicated that a short segment of 179 bp upstream of the TSP (-155 bp  upstream of the transcription start site) retained not only the promoter’s driving power, but also its tapetum-specificity. Cis-acting element search in this short segment revealed the presence of numbers of organ- and tissuespecific motifs, including pollen-specific LAT52 and SLG13. These results indicated that the  tapetumspecificity of ATA7 gene is mainly conferred by its promoter, and such a promoter, in particular, the core one should be useful both for identification of tapetum-involved genes and for biotechnological applications.Key words: Arabidopsis, Anther7 gene of Arabidopsis theliana (ATA7), anther-specific promoter, tapetumspecific promoter

Travel Reliability Inventory for Chicago

The overarching goal of this research project is to enable state DOTs to document and monitor the reliability performance of their highway networks. To this end, a computer tool, TRIC, was developed to produce travel reliability inventories from various traffic data sources. In due course, travel reliability inventories will provide necessary inputs to next-generation transportation decision-making tools that are sensitive to travel reliability. TRIC incorporates reliable routing algorithms to construct and rank travel reliability indexes on routes between any given origin-destination pair in the network. It also implements a basic graphical user interface for creating and visualizing travel reliability indexes and archiving them in ESRI shape format, which can be read by most GIS (geographic information system) software. Case studies using data from the Chicago area were conducted demonstration purposes.Illinois Department of Transportation ICT-13-011 UILU-ENG-2013-2006published or submitted for publicationnot peer reviewe

Global Proteomic Analysis of Lysine Malonylation in Toxoplasma gondii

Lysine malonylation (Kmal) is a new post-translational modification (PTM), which has been reported in several prokaryotic and eukaryotic species. Although Kmal can regulate many and diverse biological processes in various organisms, knowledge about this important PTM in the apicomplexan parasite Toxoplasma gondii is limited. In this study, we performed the first global profiling of malonylated proteins in T. gondii tachyzoites using affinity enrichment and Liquid chromatography-tandem mass spectrometry (LC-MS/MS) analysis. Three experiments performed in tandem revealed 294, 345, 352 Kmal sites on 203, 236, 230 malonylated proteins, respectively. Computational analysis showed the identified malonylated proteins to be localized in various subcellular compartments and involved in many cellular functions, particularly mitochondrial function. Additionally, one conserved Kmal motif with a strong bias for cysteine was detected. Taken together, these findings provide the first report of Kmal profile in T. gondii and should be an important resource for studying the physiological roles of Kmal in this parasite

Cigarette Smoke Extract Stimulates Rat Pulmonary Artery Smooth Muscle Cell Proliferation via PKC-PDGFB Signaling

Accumulating evidence suggests a direct role for cigarette smoke in pulmonary vascular remodeling, which contributes to the development of pulmonary hypertension. However, the molecular mechanisms underlying this process remain poorly understood. Platelet-derived growth factor (PDGF) is a potential mitogen and chemoattractant implicated in several biological processes, including cell survival, proliferation, and migration. In this study, we investigated the effect of cigarette smoke extract (CSE) on cell proliferation of rat pulmonary artery smooth muscle cells (rPASMCs). We found that stimulation of rPASMCs with CSE significantly increased cell proliferation and promoted cell cycle progression from G1 phase to the S and G2 phases. CSE treatment also significantly upregulated the mRNA and protein levels of PDGFB and PDGFRβ. Our study also revealed that Rottlerin, an inhibitor of PKCδ signaling, prevented CSE-induced cell proliferation, attenuated the increase of S and G2 phase populations induced by CSE treatment, and downregulated PDGFB and PDGFRβ mRNA and protein levels in rPASMCs exposed to CSE. Collectively, our data demonstrated that CSE-induced cell proliferation of rPASMCs involved upregulation of the PKCδ-PDGFB pathway

RHΔgra17Δnpt1 strain of Toxoplasma gondii elicits protective immunity against acute, chronic and congenital toxoplasmosis in mice

In the present study, a dense granule protein 17 (gra17) and novel putative transporter (npt1) double deletion mutant of Toxoplasma gondii RH strain was engineered. The protective efficacy of vaccination using RHΔgra17Δnpt1 tachyzoites against acute, chronic, and congenital toxoplasmosis was studied in a mouse model. Immunization using RHΔgra17Δnpt1 induced a strong humoral and cellular response, as indicated by the increased levels of anti-T. gondii specific IgG, interleukin 2 (IL-2), IL-10, IL-12, and interferon-gamma (IFN-γ). Vaccinated mice were protected against a lethal challenge dose (103 tachyzoites) of wild-type homologous (RH) strain and heterologous (PYS and TgC7) strains, as well as against 100 tissue cysts or oocysts of Pru strain. Vaccination also conferred protection against chronic infection with 10 tissue cysts or oocysts of Pru strain, where the numbers of brain cysts in the vaccinated mice were significantly reduced compared to those detected in the control (unvaccinated + infected) mice. In addition, vaccination protected against congenital infection with 10 T. gondii Pru oocysts (administered orally on day 5 of gestation) as shown by the increased litter size, survival rate and the bodyweight of pups born to vaccinated dams compared to those born to unvaccinated + infected dams. The brain cyst burden of vaccinated dams was significantly lower than that of unvaccinated dams infected with oocysts. Our data show that T. gondii RHΔgra17Δnpt1 mutant strain can protect mice against acute, chronic, and congenital toxoplasmosis by balancing inflammatory response with immunogenicity

NEUROPROTECTIVE EFFECT CORILAGIN IN SPINAL CORD INJURY RAT MODEL BY INHIBITING NUCLEAR FACTOR-KB, INFLAMMATION AND APOPTOSIS

Background: Neurological functions get altered in a patient suffering from spinal cord injury (SCI). Present study evaluates the neuroprotective effect of corilagin in spinal cord injury rats by inhibiting nuclear factor-kappa B (NF-κB), inflammatory mediators and apoptosis. Materials and method: Spinal cord injury was produced by mechanical injury to spinal cord. All the rats were treated with corilagin (10 & 15 mg/kg, ip) for the duration of 4 week. Neurological functions were evaluated in all the rats by Basso,Beattie and Brenahan scale (BBB scale). Moreover, at the end of treatment rats were sacrificed and expression of NF-κB, concentration of cytokines like Tumor necrosis factor α (TNFα) & interlukin-6 (IL6) and oxidative stress parameters like Malondialdehyde (MDA) and toal antioxidant capacity (TAC) in spinal tissue of spinal cord injured rats. Immunohistological study was also performed to assess the apoptosis of neuronal cell. Result: There were significant (

Global profiling of lysine 2-hydroxyisobutyrylome in Toxoplasma gondii using affinity purification mass spectrometry

Lysine 2-hydroxyisobutyrylation (Khib) is a recently discovered and evolutionarily conserved form of protein post-translational modification (PTM) found in mammalian and yeast cells. Previous studies have shown that Khib plays roles in the activity of gene transcription and Khib-containing proteins are closely related to the cellular metabolism. In this study, a global Khib-containing analysis using the latest databases (ToxoDB 46, 8322 sequences, downloaded on April 16, 2020) and sensitive immune-affinity enrichment coupled with liquid chromatography-tandem mass spectrometry was performed. A total of 1078 Khib modification sites across 400 Khib-containing proteins were identified in tachyzoites of Toxoplasma gondii RH strain. Bioinformatics and functional enrichment analysis showed that Khib-modified proteins were associated with various biological processes, such as ribosome, glycolysis/gluconeogenesis, and central carbon metabolism. Interestingly, many proteins of the secretory organelles (e.g., microneme, rhoptry, and dense granule) that play roles in the infection cycle of T. gondii were found to be Khib-modified, suggesting the involvement of Khib in key biological process during T. gondii infection. We also found that histone proteins, key enzymes related to cellular metabolism, and several glideosome components had Khib sites. These results expanded our understanding of the roles of Khib in T. gondii and should promote further investigations of how Khib regulates gene expression and key biological functions in T. gondii

Functional characterization of dense granule proteins in Toxoplasma gondii RH strain using CRISPR-Cas9 system

Infection with the apicomplexan protozoan parasite Toxoplasma gondii is an ongoing public health problem. The parasite's ability to invade and replicate within the host cell is dependent on many effectors, such as dense granule proteins (GRAs) released from the specialized organelle dense granules, into host cells. GRAs have emerged as important determinants of T. gondii pathogenesis. However, the functions of some GRAs remain undefined. In this study, we used CRISPR-Cas9 technique to disrupt 17 GRA genes (GRA11, GRA12 bis, GRA13, GRA14, GRA20, GRA21, GRA28-31, GRA33-38, and GRA40) in the virulent T. gondii RH strain. The CRISPR-Cas9 constructs abolished the expression of the 17 GRA genes. Functional characterization of single ΔGRA mutants was achieved in vitro using cell-based plaque assay and egress assay, and in vivo in BALB/c mice. Targeted deletion of these 17 GRA genes had no significant effect neither on the in vitro growth and egress of the mutant strains from the host cells nor on the parasite virulence in the mouse model of infection. Comparative analysis of the transcriptomics data of the 17 GRA genes suggest that GRAs may serve different functions in different genotypes and life cycle stages of the parasite. In sum, although these 17 GRAs might not be essential for RH strain growth in vitro or virulence in mice, they may have roles in other strains or parasite stages, which warrants further investigations

Diversification of mitogenomes in three sympatric Altica flea beetles (Insecta, Chrysomelidae)

The Asian flea beetles Altica cirsicola, Altica fragariae and Altica viridicyanea are broadly sympatric and morphologically highly similar but feed on distantly related host plants. They have been suggested as a model for ecological speciation stud- ies. However, their phylogeny and species limits remain uncertain. In this study, we added mitochondrial genomes from multiple individuals of each species to the grow- ing database. Phylogenetic analyses based on 15 genes showed clear interspecific divergences of A. fragariae from the other species, but A. cirsicola and A. viridi- cyanea were not distinguishable by distance‐based or tree‐based methods of species delimitation due to non‐monophyly of mitogenomes relative to the morphologically defined entities, possibly affected by interspecific introgression. This was confirmed by wider sampling of mitochondrial COX1 (58 individuals) and the second internal transcribed spacer of nuclear ribosomal RNA cluster (ITS2; 68 individuals), which showed that ITS2, but not COX1, coincided with the morphological species limits. The full mitochondrial genomes are not able to shed further light on the species status, even with the most sensitive approach based on diagnostic characters, yet the whole mitogenome is useful to get improved estimates of intra‐ and interspecific variation, not affected by the stochastic error seen in individual genes