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At least 127 records · Page 7
All sourcesNASA NTRSDOE OSTI
DOE OSTI2024

Histopathological characteristics of PRRS and expression profiles of viral receptors in the piglet immune system

Porcine reproductive and respiratory syndrome (PRRS) is a highly contagious viral disease that causes significant economic losses to the swine industry worldwide. PRRS virus (PRRSV) infection is a receptor-mediated endocytosis and replication process. The purpose of this study was to determine the localization and expression of four important PRRSV receptors in immunological organs of piglets. After piglets were infected with PRRSV, Hematoxylin and Eosin staining, immunofluorescence, and Western blot were used to perform histopathological examination and receptors distribution analysis. The results showed that PRRSV caused severe damage to the piglets’ immune organs, including atrophy of the thymus and swelling of lymph node. Histopathological lesions were mainly observed in the lung and lymph node and were characterized by interstitial pneumonia, collapsed follicles, exhaustion of germinal centers, and extensive hemorrhage. Immunofluorescence staining and Western blot results showed that the receptors of CD163 and NMHCII-A were mainly distributed in the thymus, hilar lymph nodes, and mesenteric lymph nodes. However, Sn and vimentin receptors were expressed at low levels in the immune organs of piglets. The distribution of the four receptors in the immune organs was more concentrated in the cortex but was more scattered in the medulla. Compared to the control group, the relative expression of the four receptors increased significantly in most immune organs after viral infection. In conclusion, our study examined the distribution and expression of four PRRSV receptors in immunological organs. We observed a significant increase in the expression of Sn, CD163, and vimentin following viral infection. These findings may provide potential targets for future antiviral reagent design or vaccine development.

Chen, Hong↗
DOE OSTI2021

Transcriptome and Degradome Profiling Reveals a Role of miR530 in the Circadian Regulation of Gene Expression in Kalanchoë marnieriana

Crassulacean acid metabolism (CAM) is an important photosynthetic pathway for plant adaptation to dry environments. CAM plants feature a coordinated interaction between mesophyll and epidermis functions that involves refined regulations of gene expression. Plant microRNAs (miRNAs) are crucial post-transcription regulators of gene expression, however, their roles underlying the CAM pathway remain poorly investigated. Here, we present a study characterizing the expression of miRNAs in an obligate CAM species Kalanchoë marnieriana. Through sequencing of transcriptome and degradome in mesophyll and epidermal tissues under the drought treatments, we identified differentially expressed miRNAs that were potentially involved in the regulation of CAM. In total, we obtained 84 miRNA genes, and eight of them were determined to be Kalanchoë-specific miRNAs. It is widely accepted that CAM pathway is regulated by circadian clock. We showed that miR530 was substantially downregulated in epidermal peels under drought conditions; miR530 targeted two tandem zinc knuckle/PLU3 domain encoding genes (TZPs) that were potentially involved in light signaling and circadian clock pathways. Our work suggests that the miR530-TZPs module might play a role of regulating CAM-related gene expression in Kalanchoë.

Kalanchoë↗
DOE OSTI2022

Analyzing BMP2, FGFR, and TGF Beta Expressions in High-Grade Osteosarcoma Untreated and Treated Autografts Using Proteomic Analysis

In the last few decades, biological reconstruction techniques have improved greatly for treating high-grade osteosarcoma patients. To conserve the limb, and its function the affected tumor-bearing bones have been treated using liquid nitrogen and irradiation processes that enable the removal of entire tumors from the bone, and these treated autografts can be reconstructed for the patients. Here, we focus on the expressions of the growth factor family proteins from the untreated and treated autografts that play a crucial role in bone union, remodeling, and regeneration. In this proteomic study, we identify several important cytoskeletal, transcriptional, and growth factor family proteins that showed substantially low levels in untreated autografts. Interestingly, these protein expressions were elevated after treating the tumor-bearing bones using liquid nitrogen and irradiation. Therefore, from our preliminary findings, we chose to determine the expressions of BMP2, TGF-Beta, and FGFR proteins by the target proteomics approach. Using a newly recruited validation set, we successfully validate the expressions of the selected proteins. Furthermore, the increased growth factor protein expression after treatment with liquid nitrogen may contribute to bone regeneration healing, assist in faster recovery, and reduce local recurrence and metastatic spread in high-grade sarcoma patients.

60 APPLIED LIFE SCIENCES↗
DOE OSTI2025

Astrocyte FABP7 Modulates Seizure Activity-Dependent Protein Expression in Mouse Brain

Background/Objectives: Patients with epilepsy commonly experience patterns of seizures that change with sleep/wake behavior or diurnal rhythms. The cellular and molecular mechanisms that underlie these patterns in seizure activity are not well understood but may involve non-neuronal cells, such as astrocytes. Our previous studies show the critical importance of one specific astrocyte factor, the brain-type fatty acid binding protein Fabp7, in the regulation of time-of-day-dependent electroshock seizure threshold and neural activity-dependent gene expression in mice. Here, we examined whether Fabp7 influences differential seizure activity-dependent protein expression, by comparing Fabp7 knockout (KO) to wild-type (WT) mice under control conditions and after reaching the maximal electroshock seizure threshold (MEST). Methods: We analyzed the proteome in cortical–hippocampal extracts from MEST and SHAM groups of WT and KO mice using mass spectrometry (MS), followed by Gene Ontology (GO) and pathway analyses. GO and pathway analyses of all groups revealed a diverse set of up- and downregulated differentially expressed proteins (DEPs). Results: We identified 65 significant DEPs in the comparison of KO SHAM versus WT SHAM; 33 proteins were upregulated and 32 were downregulated. We found downregulation in mitochondrial-associated proteins in WT MEST compared to WT SHAM controls, including Slc1a4, Slc25a27, Cox7a2, Cox8a, Micos10, and Atp5mk. Several upregulated DEPs in the KO SHAM versus WT SHAM comparison were associated with the 20S proteasomal subunit, suggesting proteasomal activity is elevated in the absence of Fabp7 expression. We also observed 92 DEPs significantly altered in the KO MEST versus WT MEST, with 49 proteins upregulated and 43 downregulated. Conclusions: Together, these data suggest that the astrocyte Fabp7 regulation of time-of-day-mediated neural excitability is modulated by multiple cellular mechanisms, which include proteasomal pathways, independent of its role in activity-dependent gene expression.

Neural Excitability↗
DOE OSTI2021

Gamete expression of TALE class HD genes activates the diploid sporophyte program in Marchantia polymorpha

Eukaryotic life cycles alternate between haploid and diploid phases and in phylogenetically diverse unicellular eukaryotes, expression of paralogous homeodomain genes in gametes primes the haploid-to-diploid transition. In the unicellular chlorophyte alga Chlamydomonas, KNOX and BELL TALE-homeodomain genes mediate this transition. We demonstrate that in the liverwort Marchantia polymorpha, paternal (sperm) expression of three of five phylogenetically diverse BELL genes, MpBELL234, and maternal (egg) expression of both MpKNOX1 and MpBELL34 mediate the haploid-to-diploid transition. Loss-of-function alleles of MpKNOX1 result in zygotic arrest, whereas a loss of either maternal or paternal MpBELL234 results in variable zygotic and early embryonic arrest. Expression of MpKNOX1 and MpBELL34 during diploid sporophyte development is consistent with a later role for these genes in patterning the sporophyte. These results indicate that the ancestral mechanism to activate diploid gene expression was retained in early diverging land plants and subsequently co-opted during evolution of the diploid sporophyte body.

59 BASIC BIOLOGICAL SCIENCES↗
DOE OSTI2021

Expression of dicarboxylate transporter from setaria italica in transgenic plants to increase yield

Compositions and methods for increasing plant growth for higher crop yield are provided. The methods involve the expression in a plant of interest of at least one C4 transporter coding sequence. Plants showing increased expression of one or more C4 transporter coding sequence of interest are encompassed by the invention. It is recognized that any method for increasing the expression of the C4 transporter coding sequences in a plant of interest can be used in the practice of the methods disclosed herein. Such methods include transformation, breeding and the like. Increased expression of the C4 transporter coding sequences in the plant of interest results in yield gains. Expression cassettes and vectors comprising the C4 transporter sequences disclosed herein are also provided herein. Methods for identifying genes under positive selection in plants that use C4 photosynthesis are disclosed and provided herein.

Brutnell, Thomas P.↗
DOE OSTI2024

Multiplex characterization of microbial traits using dual barcoded nucleic acid fragment expression library

Disclosed herein are barcoded expression libraries comprising a plurality of expression vectors, wherein each expression vector comprises a nucleic acid fragment flanked by a first barcode and a second barcode. Further disclosed herein are methods of making the barcoded expression libraries and methods of conducting functional analysis using the barcoded expression libraries.

Mutalik, Vivek K.↗
DOE OSTI2022

High-yield recombinant bacterial expression of 13 C-, 15 N-labeled, serine-16 phosphorylated, murine amelogenin using a modified third generation genetic code expansion protocol

Amelogenin constitutes ~ 90% of the enamel matrix in the secretory stage of amelogenesis, a still poorly understood process that results in the formation of the hardest and most mineralized tissue in vertebrates – enamel. Most biophysical research with amelogenin uses recombinant protein expressed in Escherichia coli. In addition to providing copious amounts of protein, recombinant expression allows 13 C- and 15 N-labelling for detailed structural studies using NMR spectroscopy. However, native amelogenin is phosphorylated at one position, Ser-16 in murine amelogenin, and a lingering question overshadowing decades of enamel research with recombinant amelogenin is the effect of phosphorylation on its biophysical properties. Using a modified genetic code expansion protocol we have expressed and purified uniformly 13 C-, 15 N-labeled murine amelogenin (pS16M179) with ~95% of the protein being correctly phosphorylated. Homogeneous phosphorylation was achieved using commercially available, enriched, 13 C-, 15 N-labeled media and protein expression was induced with isopropyl β-D-1-thiogalactopyranoside at 310 K. Phosphoserine incorporation was verified from one-dimensional 31P NMR spectra, comparison of 1 H- 15 N HSQC spectra, Phos-tag SDS PAGE, and mass spectrometry. Phosphorus-31 NMR spectra for pS16M179 under conditions known to trigger amelogenin self-assembly into nanospheres confirm nanosphere models with buried N-termini. Lambda phosphatase treatment of these nanospheres results in the dephosphorylation of pS16M179, confirming that smaller oligomers and monomers with exposed N-termini are in equilibrium with nanospheres. Such 13 C-, 15 N-labeling of amelogenin with accurately encoded phosphoserine incorporation will accelerate biomineralization research to understand amelogenesis and stimulate the expanded use of genetic code expansion protocols to introduce phosphorylated amino acids into proteins.

31P-NMR↗
DOE OSTI2026

A Fluorescence‐Based Transient Expression Assay for the Analysis of Upstream Open Reading Frames in Plants

Upstream open reading frames (uORFs) are regulatory elements present in the 5′ leaders of mRNA that can significantly impact downstream gene expression in eukaryotes. In crop engineering, editing of uORFs can provide an avenue to upregulate expression of native genes without the need to add persistent transgenic copies. Even with genome-wide methods to identify translated uORFs such as ribosome profiling, their functional characterization depends on validation through reporter gene assays and mutagenesis studies. Current screening methods for plants use luciferases or protoplasts to measure differential gene expression between wild-type and mutated transcript leaders, which requires tissue processing and/or substrate addition. Here, we present a time- and cost-efficient alternative to investigate transcript leaders by co-expression of two fluorescent proteins in Nicotiana benthamiana leaf tissue and test our assay on genes involved in photoprotection, editing of which could provide a pathway to increase CO 2 assimilation during sun–shade transitions.

Nicotiana benthamiana↗
DOE OSTI2025

Can protein expression be ‘solved’?

Recombinant protein expression is central to biotechnology’s application in academic exploration as well as human health, climate applications and the bioeconomy in general. However, not all proteins can be expressed in all organisms, and the field lacks a predictive model of soluble protein overexpression that could replace laborious experimental trial-and-error. Here, we discuss the state of the field and identify the lack of large, high-fidelity datasets as the primary bottleneck to progress. We review possible assays that could be used for data collection to identify a path toward an extensible experimental platform for collecting soluble recombinant protein overexpression data across organisms. We suggest that the resulting dataset should be used to train increasingly generalizable predictive models of protein expression to answer the question: “How can predictive protein expression be solved?”.

59 BASIC BIOLOGICAL SCIENCES↗
DOE OSTI2024

Metabolic flux optimization of iterative pathways through orthogonal gene expression control: Application to the β-oxidation reversal

Balancing relative expression of pathway genes to minimize flux bottlenecks and metabolic burden is one of the key challenges in metabolic engineering. This is especially relevant for iterative pathways, such as reverse β-oxidation (rBOX) pathway, which require control of flux partition at multiple nodes to achieve efficient synthesis of target products. Here, we develop a plasmid-based inducible system for orthogonal control of gene expression (referred to as the TriO system) and demonstrate its utility in the rBOX pathway. Leveraging effortless construction of TriO vectors in a plug-and-play manner, we simultaneously explored the solution space for enzyme choice and relative expression levels. Remarkably, varying individual expression levels led to substantial change in product specificity ranging from no production to optimal performance of about 90% of the theoretical yield of the desired products. We obtained titers of 6.3 g/L butyrate, 2.2 g/L butanol and 4.0 g/L hexanoate from glycerol in E. coli, which exceed the best titers previously reported using equivalent enzyme combinations. Since a similar system behavior was observed with alternative termination routes and higher-order iterations, we envision our approach to be broadly applicable to other iterative pathways besides the rBOX. Here, considering that high throughput, automated strain construction using combinatorial promoter and RBS libraries remain out of reach for many researchers, especially in academia, tools like the TriO system could democratize the testing and evaluation of pathway designs by reducing cost, time and infrastructure requirements.

59 BASIC BIOLOGICAL SCIENCES↗
DOE OSTI2020

Logic-based analysis of gene expression data predicts association between TNF, TGFB1 and EGF pathways in basal-like breast cancer

For breast cancer, clinically important subtypes are well characterized at the molecular level in terms of gene expression profiles. In addition, signaling pathways in breast cancer have been extensively studied as therapeutic targets due to their roles in tumor growth and metastasis. However, it is challenging to put signaling pathways and gene expression profiles together to characterize biological mechanisms of breast cancer subtypes since many signaling events result from post-translational modifications, rather than gene expression differences. We designed a logic-based computational framework to explain the differences in gene expression profiles among breast cancer subtypes using Pathway Logic and transcriptional network information. Pathway Logic is a rewriting-logic-based formal system for modeling biological pathways including post-translational modifications. Our method demonstrated its utility by constructing subtype-specific path from key receptors (TNFR, TGFBR1 and EGFR) to key transcription factor (TF) regulators (RELA, ATF2, SMAD3 and ELK1) and identifying potential association between pathways via TFs in basal-specific paths, which could provide a novel insight on aggressive breast cancer subtypes. Lastly, codes and results are available at http://epigenomics.snu.ac.kr/PL/.

59 BASIC BIOLOGICAL SCIENCES↗
DOE OSTI2023

A Suite of Constitutive Promoters for Tuning Gene Expression in Plants

The need for convenient tools to express transgenes over a large dynamic range is pervasive throughout plant synthetic biology; however, current efforts are largely limited by the heavy reliance on a small set of strong promoters, precluding more nuanced and refined engineering endeavors in planta . To address this technical gap, we characterize a suite of constitutive promoters that span a wide range of transcriptional levels and develop a GoldenGate-based plasmid toolkit named PCONS, optimized for versatile cloning and rapid testing of transgene expression at varying strengths. We demonstrate how easy access to a stepwise gradient of expression levels can be used for optimizing synthetic transcriptional systems and the production of small molecules in planta . We also systematically investigate the potential of using PCONS as an internal standard in plant biology experimental design, establishing the best practices for signal normalization in experiments. Although our library has primarily been developed for optimizing expression in N. benthamiana , we demonstrate the translatability of our promoters across distantly related species using a multiplexed reporter assay with barcoded transcripts. In conclusion, our findings showcase the advantages of the PCONS library as an invaluable toolkit for plant synthetic biology.

59 BASIC BIOLOGICAL SCIENCES↗
DOE OSTI2022

Exploring Camelina sativa lipid metabolism regulation by combining gene co-expression and DNA affinity purification analyses

Camelina (Camelina sativa) is an annual oilseed plant that is gaining momentum as a biofuel cover crop. Understanding gene regulatory networks is essential to deciphering plant metabolic pathways, including lipid metabolism. Furthermore, we take advantage of a growing collection of gene expression datasets to predict transcription factors (TFs) associated with the control of Camelina lipid metabolism. We identified approximately 350 TFs highly co-expressed with lipid-related genes (LRGs). These TFs are highly represented in the MYB, AP2/ERF, bZIP, and bHLH families, including a significant number of homologs of well-known Arabidopsis lipid and seed developmental regulators. After prioritizing the top 22 TFs for further validation, we identified DNA-binding sites and predicted target genes for 16 out of the 22 TFs tested using DNA affinity purification followed by sequencing (DAP-seq). Enrichment analyses of targets supported the co-expression prediction for most TF candidates, and the comparison to Arabidopsis revealed some common themes, but also aspects unique to Camelina. Within the top potential lipid regulators, we identified CsaMYB1, CsaABI3AVP1-2, CsaHB1, CsaNAC2, CsaMYB3, and CsaNAC1 as likely involved in the control of seed fatty acid elongation and CsaABI3AVP1-2 and CsabZIP1 as potential regulators of the synthesis and degradation of triacylglycerols (TAGs), respectively. Altogether, the integration of co-expression data and DNA-binding assays permitted us to generate a high-confidence and short list of Camelina TFs involved in the control of lipid metabolism during seed development.

59 BASIC BIOLOGICAL SCIENCES↗
DOE OSTI2023

Rhizosphere and detritusphere habitats modulate expression of soil N-cycling genes during plant development

Interactions between plant roots and rhizosphere bacteria modulate nitrogen (N)-cycling processes and create habitats rich in low molecular weight compounds (exudates) and complex organic molecules (decaying root litter) compared to those of bulk soil. Microbial N-cycling is regulated by edaphic conditions and genes from many interconnected metabolic pathways, but most studies of soil N-cycling gene expression have focused on single pathways. Currently, we lack a comprehensive understanding of the interplay between soil N-cycling gene regulation, spatial habitat, and time. We present results from a replicated time series of soil metatranscriptomes; we followed gene expression of multiple N transformations in four soil habitats (rhizosphere, detritusphere, rhizo-detritusphere, and bulk soil) during active root growth for the annual grass, Avena fatua. The presence of root litter and living roots significantly altered the trajectories of N-cycling gene expression. Upregulation of assimilatory nitrate reduction in the rhizosphere suggests that rhizosphere bacteria were actively competing with roots for nitrate. Simultaneously, ammonium assimilatory pathways were upregulated in both rhizosphere and detritusphere soil, which could have limited N availability to plants. The detritusphere supported dissimilatory processes DNRA and denitrification. Expression of nitrification genes was dominated by three phylotypes of Thaumarch aeota and was upregulated in bulk soil. Unidirectional ammonium assimilation and its regulatory genes (GS/GOGAT) were upregulated near relatively young roots and highly decayed root litter, suggesting N may have been limiting in these habitats (GS/GOGAT is typically activated under N limitation). In conclusion, our comprehensive analysis indicates that differences in carbon and inorganic N availability control contemporaneous transcription of N-cycling pathways in soil habitats.

54 ENVIRONMENTAL SCIENCES↗
DOE OSTI2022

Transcriptome Sequencing of Agave angustifolia Reveals Conservation and Diversification in the Expression of Cinnamyl Alcohol Dehydrogenase Genes in Agave Species

Agave angustifolia is an important crassulacean acid metabolism plant species, with wide applications in beverage and sisal fiber production. In this study, we carried out a transcriptome analysis of A. angustifolia leaves, generating a total of 58,482,436 clean reads through Illumina paired-end sequencing. De novo transcriptome assembly generated 67,314 unigenes, with about half of them having homologs in four public databases. In the Nr database, Asparagus officinalis was shown to be most closely related to agave, and the early angiosperm Amborella trichopoda was selected as an outgroup species. We further identified five, two, seven, seven, seven, six and six CAD genes in asparagus, amborella, A. deserti, A. tequilana, A. americana, A. hybrid H11648 and A. angustifolia, respectively. The maximum likelihood phylogenetic tree revealed the species-specific expansion of CAD genes in arabidopsis, rice and agave. The expression analysis indicated the conservatively expressed CAD1/2/4/6, providing candidate targets for manipulation to improve lignin traits. The species-specific expression of CAD3/5/7 indicates the existence of different regulatory mechanisms controlling the expression of these genes in agave species. This study presents the first transcriptome dataset of A. angustifolia, facilitating future studies on lignin biosynthesis in agave.

59 BASIC BIOLOGICAL SCIENCES↗
DOE OSTI2021

Pulmonary ACE2 expression in neonatal and adult rats

Children show a distinct presentation of COVID‐19, characterized by a lower incidence and mild phenotype, but the reason for this is still unknown. The angiotensin‐converting enzyme 2 (ACE2) functions as the primary cell entry receptor for Severe acute respiratory syndrome coronavirus 2 (SARS‐CoV‐2) and is thought to cause distinct clinical features between children and old people. The primary purpose of this study was to determine whether differences exist in the level of expression and distribution of ACE2 between neonatal and adult rat lungs. The lung tissues from rats of various ages were used to investigate the expression patterns of ACE2. Western blot, immunohistochemistry, and immunofluorescence were used to quantify or identify the localization of ACE2 in rat lungs. ACE2 was homogenously expressed in fewer alveolar type II (AT2) cells in the neonatal lung, with no polarization to the alveolar space and additional expression in pulmonary endothelium when compared to adult rat lungs. These findings suggest that the patterns of ACE2 distribution and cellular localization in rat lungs change with age.

Zhao, Depeng↗
DOE OSTI2020

Shade signals alter the expression of circadian clock genes in newly-formed bioenergy sorghum internodes

Stem internodes of bioenergy sorghum inbred R.07020 are longer at high plant density (shade) than at low plant density (control). Initially, the youngest newly-formed subapical stem internodes of shade-treated and control plants are comparable in length. However, full-length internodes of shade-treated plants are three times longer than the internodes of the control plants. To identify the early molecular events associated with internode elongation in response to shade, we analyzed the transcriptome of the newly-formed internodes of shade-treated and control plants sampled between 4 and 6 hr after the start of the light period (14 hr light/10 hr dark). Sorghum genes homologous to the Arabidopsis shade marker genes ATHB2 and PIL1 were not differentially expressed. The results indicate that shade signals promote internode elongation indirectly because sorghum internodes are not illuminated and grow while enclosed with leaf sheaths. Sorghum genes homologous to the Arabidopsis morning-phased circadian clock genes LHY, RVE, and LNK were downregulated and evening-phased genes such as TOC1, PRR5, and GI were upregulated in young internodes in response to shade. We hypothesize that a change in the function or patterns of expression of the circadian clock genes is the earliest molecular event associated with internode elongation in response to shade in bioenergy sorghum. Increased expression of CycD1, which promotes cell division, and decreased expression of cell wall-loosening and MBF1-like genes, which promote cell expansion, suggest that shade signals promote internode elongation in bioenergy sorghum in part through increasing cell number by delaying transition from cell division to cell expansion.

54 ENVIRONMENTAL SCIENCES↗