EGFP Fusion Protein Research Articles

Design of the conserved epitope peptide of SARS-CoV-2 spike protein as the broad-spectrum COVID-19 vaccine

Our previous study has found that monoclonal antibodies targeting a conserved epitope peptide spanning from residues 1144 to 1156 of SARS-CoV-2 spike (S) protein, namely S(1144–1156), can broadly neutralize all of the prevalent SARS-CoV-2 strains, including the wild type, Alpha, Epsilon, Delta, and Gamma variants. In the study, S(1144–1156) was conjugated with bovine serum albumin (BSA) and formulated with Montanide ISA 51 adjuvant for inoculation in BALB/c mice to study its potential as a vaccine candidate. Results showed that the titers of S protein-specific IgGs and the neutralizing antibodies in mouse sera against various SARS-CoV-2 variants, including the Omicron sublineages, were largely induced along with three doses of immunization. The significant release of IFN-γ and IL-2 was also observed by ELISpot assays through stimulating vaccinated mouse splenocytes with the S(1144–1156) peptide. Furthermore, the vaccination of the S(1143–1157)- and S(1142–1158)-EGFP fusion proteins can elicit more SARS-CoV-2 neutralizing antibodies in mouse sera than the S(1144–1156)-EGFP fusion protein. Interestingly, the antisera collected from mice inoculated with the S(1144–1156) peptide vaccine exhibited better efficacy for neutralizing Omicron BA.2.86 and JN.1 subvariants than Omicron BA.1, BA.2, and XBB subvariants. Since the amino acid sequences of the S(1144–1156) are highly conserved among various SARS-CoV-2 variants, the immunogen containing the S(1144–1156) core epitope can be designed as a broadly effective COVID-19 vaccine.Key points• Inoculation of mice with the S(1144–1156) peptide vaccine can induce bnAbs against various SARS-CoV-2 variants.• The S(1144–1156) peptide stimulated significant release of IFN-γ and IL-2 in vaccinated mouse splenocytes.• The S(1143–1157) and S(1142–1158) peptide vaccines can elicit more SARS-CoV-2 nAbs in mice.

Pyruvate orthophosphate dikinase is required for the acclimation to high bicarbonate concentrations in Phaeodactylum tricornutum

The model marine diatom P. tricornutum possesses efficient CO2-concentrating mechanisms (CCMs) involving the bicarbonate transport and the rapid inter-conversion of HCO3− and CO2, and it has a complete set of enzymes essential for biochemical CCM C4 pathway. However, the role of potential C4 pathway in the diatom is still controversial. Based on the analysis of previously published proteome and transcriptome data in P. tricornutum, the expression of pyruvate orthophosphate dikinase (PPDK), a key enzyme generating the primary acceptor for bicarbonate fixation in C4 pathway, was upregulated in long-term high CO2-selected populations. In our study, P. tricornutum PPDK (PtPPDK) transcript abundance was higher with short-term treatment of high NaHCO3 concentrations and overexpression of PtPPDK resulted in a cell density increase of 5–16 % from day 2 to 8. Knockdown (KD) or knockout of PtPPDK inhibited the growth and the maximal photosystem II electron transport rate, and the inhibited growth became more and more evident with the increasing NaHCO3 concentration from 1 to 8 mM in PtPPDK KD lines. Furthermore, elevating NaHCO3 from 1 to 16 mM dramatically decreased the cell density both in wild-type and PtPPDK mutants, suggesting that 1 mM NaHCO3 is enough for the photosynthesis carbon assimilation. PtPPDK was localized in the plastid stroma by eGFP fusion protein analysis at 2 mM NaHCO3, while PtPPDK-eGFP fluorescence was observed both in the stroma and periplasmic space at higher NaHCO3. It is suggested that PPDK is necessary for consuming excessive inorganic carbon (Ci) both from the plastid and extracellular environment to maintain pH homeostasis, indicating a potential function of adapting to high Ci concentrations.

Superior protective effects of PGE2 priming mesenchymal stem cells against LPS-induced acute lung injury (ALI) through macrophage immunomodulation

BackgroundMesenchymal stem cells (MSCs) have demonstrated remarkable therapeutic promise for acute lung injury (ALI) and its severe form, acute respiratory distress syndrome (ARDS). MSC secretomes contain various immunoregulatory mediators that modulate both innate and adaptive immune responses. Priming MSCs has been widely considered to boost their therapeutic efficacy for a variety of diseases. Prostaglandin E2 (PGE2) plays a vital role in physiological processes that mediate the regeneration of injured organs.MethodsThis work utilized PGE2 to prime MSCs and investigated their therapeutic potential in ALI models. MSCs were obtained from human placental tissue. MSCs were transduced with firefly luciferase (Fluc)/eGFP fusion protein for real-time monitoring of MSC migration. Comprehensive genomic analyses explored the therapeutic effects and molecular mechanisms of PGE2-primed MSCs in LPS-induced ALI models.ResultsOur results demonstrated that PGE2-MSCs effectively ameliorated lung injury and decreased total cell numbers, neutrophils, macrophages, and protein levels in bronchoalveolar lavage fluid (BALF). Meanwhile, treating ALI mice with PGE2-MSCs dramatically reduced histopathological changes and proinflammatory cytokines while increasing anti-inflammatory cytokines. Furthermore, our findings supported that PGE2 priming improved the therapeutic efficacy of MSCs through M2 macrophage polarization.ConclusionPGE2-MSC therapy significantly reduced the severity of LPS-induced ALI in mice by modulating macrophage polarization and cytokine production. This strategy boosts the therapeutic efficacy of MSCs in cell-based ALI therapy.

Doxycycline-inducible Expression of Proteins at Near-endogenous Levels in Mammalian Cells Using the Sleeping Beauty Transposon System.

The function of a protein within a cell critically depends on its interaction with other proteins as well as its subcellular localization. The expression of mutants of a particular protein that have selective perturbation of specific protein interaction motifs is a very useful strategy for resolving a protein's mechanism of action in a cellular process. In addition, expression of fluorescent protein fusions is a key strategy for determining the subcellular localization of a protein. These strategies require tight regulation to avoid potential alterations in protein interactions or localizations that can result from protein overexpression. Previous work led to the development of a Sleeping Beauty transposon system that allows doxycycline-inducible expression of protein mutants or fusions; titration of doxycycline allows expression of protein fusions or mutants at near endogenous levels. When used in combination with siRNA gene silencing, this strategy allows for knockdown-rescue experiments to assess the function of specific protein mutants. In this protocol, we describe the use of this Sleeping Beauty strategy for expression of eGFP fusion or mutant proteins in ARPE-19 and MDA-MB-231 cells. This includes design of expression plasmids, transfection, and selection to obtain stable engineered cells, as well as doxycycline treatment for controlled induction of protein expression, either alone or in combination with siRNA silencing for knockdown-rescue experiments. This strategy is advantageous as it allows rapid generation of stable cells for controlled protein expression, suitable for functional studies that require knockdown-rescue as well as various forms of live cell fluorescence imaging. Key features • Highly versatile doxycycline-inducible expression system that can be used in various mammalian cell lines. • Stable integration of transgene allows for sustained and stable expression. • Titration of doxycycline levels allows expression of transgene at near endogenous levels.

Identification and Functional Analysis of Acyl-Acyl Carrier Protein Δ9 Desaturase from Nannochloropsis oceanica.

The oleaginous marine microalga Nannochloropsis oceanica strain IMET1 has attracted increasing attention as a promising photosynthetic cell factory due to its unique excellent capacity to accumulate large amounts of triacylglycerols and eicosapentaenoic acid. To complete the genomic annotation for genes in the fatty acid biosynthesis pathway of N. oceanica, we conducted the present study to identify a novel candidate gene encoding the archetypical chloroplast stromal acyl-acyl carrier protein Δ9 desaturase. The full-length cDNA was generated using rapid-amplification of cDNA ends, and the structure of the coding region interrupted by four introns was determined. The RT-qPCR results demonstrated the upregulated transcriptional abundance of this gene under nitrogen starvation condition. Fluorescence localization studies using EGFP-fused protein revealed that the translated protein was localized in chloroplast stroma. The catalytic activity of the translated protein was characterized by inducible expression in Escherichia coli and a mutant yeast strain BY4389, indicating its potential desaturated capacity for palmitoyl-ACP (C16:0-ACP) and stearoyl-ACP (C18:0-ACP). Further functional complementation assay using BY4839 on plate demonstrated that the expressed enzyme restored the biosynthesis of oleic acid. These results support the desaturated activity of the expressed protein in chloroplast stroma to fulfill the biosynthesis and accumulation of monounsaturated fatty acids in N. oceanica strain IMET1.

Modulation of Rab GDP-Dissociation Inhibitor Trafficking and Expression by the Transmembrane Protein 59 (TMEM59)

Transmembrane protein 59 (TMEM59) is a type I transmembrane protein. However, the characterization and functions of TMEM59 in cells are not clear. Our results showed that TMEM59 localizes to vesicular structures. Further co-localization studies illustrated that TMEM59 is mainly distributed in the lysosome and acidic vesicular. TMEM59 movement between the nucleus and cell membrane was observed in living cells expressing TMEM59–EGFP fusion proteins. In addition, cell surface transport of amyloid precursor protein (APP) was significantly inhibited by TMEM59 and increased APP levels in HEK296T cells. TMEM59 also significantly inhibits transport of Rab GDP dissociation inhibitor alpha (GDI1) and Rab GDP dissociation inhibitor beta (GDI2), and further increases expression of GDI1 and GDI2 proteins in the cytoplasm. However, TMEM59 does not affect protein expression and localization of BACE2. These results suggest that TMEM59 may be involved in the packaging of acidic vesicles, modulated transport, and processing of APP, GDI1, and GDI2.

PRG3 and PRG5 C-Termini: Important Players in Early Neuronal Differentiation

The functional importance of neuronal differentiation of the transmembrane proteins' plasticity-related genes 3 (PRG3) and 5 (PRG5) has been shown. Although their sequence is closely related, they promote different morphological changes in neurons. PRG3 was shown to promote neuritogenesis in primary neurons; PRG5 contributes to spine induction in immature neurons and the regulation of spine density and morphology in mature neurons. Both exhibit intracellularly located C-termini of less than 50 amino acids. Varying C-termini suggested that these domains shape neuronal morphology differently. We generated mutant EGFP-fusion proteins in which the C-termini were either swapped between PRG3 and PRG5, deleted, or fused to another family member, plasticity-related gene 4 (PRG4), that was recently shown to be expressed in different brain regions. We subsequently analyzed the influence of overexpression in immature neurons. Our results point to a critical role of the PRG3 and PRG5 C-termini in shaping early neuronal morphology. However, the results suggest that the C-terminus alone might not be sufficient for promoting the morphological effects induced by PRG3 and PRG5.

A Perilipin Affects Lipid Droplet Homeostasis and Aerial Hyphal Growth, but Has Only Small Effects on Virulence in the Insect Pathogenic Fungus Beauveria bassiana.

Lipid assimilation, storage, and turnover impact growth, development, and virulence in many microbial pathogens including fungi. Perilipins are proteins associated with lipid droplets (LDs) that mediate their assembly and turnover. Here, we characterized the Beauveria bassiana (BbPlin1) perilipin. BbPlin1 expression was higher in minimal media than in rich media, and, using a BbPlin1::eGFP fusion protein, the protein was shown to be co–localized to LDs, with the high expression seen during infection and proliferation within the insect (Galleria mellonella) host that dramatically decreased to almost no expression during fungal outgrowth on cadavers including in conidia, but that BbPlin1 production resumed in the conidia once placed in nutrient–containing media allowing for germination and growth. Characterization of a targeted gene deletion strain (ΔBbPlin1) revealed a dramatic (>30%) reduction in cellular LD content, promotion of aerial hyphal growth, and a small decrease in virulence, with little to no effects on vegetative growth and stress responses. However, in the ΔBbPlin1 strain, expression of the complementary LD–associated caleosin gene, BbCal1, was enhanced under nutrient–poor conditions, although no changes in BbPlin1 expression were seen in a ΔBbCal1 strain and the expression of BbPlin1 in the ΔBbCal1 strain did not change LD patterns in cells. Transcriptome and RT–PCR analyses indicated increased expression of lipid metabolism–related genes, including triacylglyercol lipase 3, enoyl–CoA isomerase, and diacylglycerol–O–acetyl transferase in the BbPlin1 deletion mutant. Lipid profile analyses confirmed that the loss of BbPlin1 significantly reduced the cellular levels of contents of triacylglycerol, diacylglycerol, and phosphatidylethanolamine as compared to the wild–type strain. These results demonstrate the involvement of the B. bassiana perilipin in mediating lipid homeostasis, fungal aerial hyphal growth, and virulence, revealing critical cycling from high expression during nutrient utilization within host cadavers to low expression during growth on the surface of the cadaver during the infection process.

FC076: Development of a High-Throughput in Vivo Drug Screening Assay Using a FSGS-Like Zebrafish Model

Abstract BACKGROUND AND AIMS Focal segmental sclerosis (FSGS) is a severe histopathological condition which often results in end-stage renal disease. Loss of podocytes and severe changes of podocyte foot process morphology accompanied by proteinuria are the hallmarks of FSGS. Since no therapeutic agents are available until now, further efforts must be made to identify new drugs to treat this disease. The zebrafish larva is a widely used kidney research model as the morphology and function of zebrafish and mammalian glomeruli are very similar. Since our group has already established a FSGS-like zebrafish injury model based on the nitroreductase-metronidazole cell ablation system, we have developed a high-throughput based in vivo drug screening assay. METHOD We used a zebrafish screening strain that expresses mCherry and the bacterial enzyme nitroreductase in podocytes. Additionally, a circulating vitamin-D-binding eGFP fusion protein with a size of 78 kDa is expressed and serves as a read-out for proteinuria. Incubation with 80 µM metronidazole for 24 h at 4 days post-fertilization (dpf) induces an FSGS-like phenotype in these larvae. A library of 138 epigenetic drugs were screened and the results are presented herein. Groups of 12 larvae received injury induction as well as drug treatment and were transferred individually into 96-well plates prepared with custom agarose molds. Larvae were oriented laterally and imaged with a high-content screening microscope (Acquifer Imaging Machine). The eGFP signal in the caudal vasculature as well as the glomerular mCherry signal served as readout systems. At 6 dpf, the same larvae and regions were imaged again. Custom written FIJI codes automatically segmented and measured the fluorescence intensities of both readouts and timepoints. For each larva, intensity ratios were determined and the ratios of each treatment group served as the input for statistical analysis. RESULTS Treatment for 24 hours with 80 µM metronidazole resulted in a measurable decline of the eGFP signal in the vasculature and of the podocyte mCherry signal compared to the vehicle (DMSO) group. Simultaneous treatment of metronidazole with the 138 drugs in the epigenetics library resulted in beneficial outcomes in varying degrees (9 drugs were positive, 36 compounds were lethal). CONCLUSION This new in vivo semi-automated high-throughput compatible drug screening assay allows rapid identification of FSGS protective drugs. Here we screened 138 epigenetic drugs, which led to the identification of 9 potential drugs which have had a beneficial effect on podocytes and/or the glomerular filtration barrier.

Myeloid cell interleukin-10 is essential for atherosclerosis protection in Apoe−/− mice.

Abstract Atherosclerosis, the disease underlying stroke, myocardial infarction and ischemic heart failure, is initiated by the subendothelial retention of lipoproteins and cholesterol, which then triggers a non-resolving inflammatory process driving plaque progression in the artery wall. Myeloid cells and in particular macrophages are the primary driver of this process. IL10 is a prototypic anti-inflammatory cytokine made by several immune cells such as macrophages. IL10 is also essential for the protection against atherosclerosis. However, it is not known which cells are responsible for the IL10 production mediating protection from atherosclerosis. Here, we crossed the VertX mouse model, in which an IRES-enhanced green fluorescent protein (eGFP) fusion protein was placed downstream of exon 5 of the IL-10 gene, to the atherosclerosis-prone Apoe−/− background. We found that myeloid cells express high levels of IL10 GFP in VertX-Apoe−/− mice. By scRNAseq we found that macrophages especially inflammatory macrophages in plaques are the main producers of IL10 in atherosclerosis. To address whether IL10 secreted by myeloid cells is essential for the protection, we developed a LyzMCreIL-10flox/flox mouse crossed in the Apoe−/− background and confirmed that macrophages were unable to secrete IL-10. We discovered that the LyzMCreIL-10flox/floxApoe−/− mice developed significantly more atherosclerosis than LyzMCreIL-10+/+Apoe−/−, both on chow and lipid-rich diet. Flow cytometry data and cytokine measurements suggest that the depletion of IL-10 in myeloid cells increases Th17 cells with elevated IL-17a, IL-17f and CCL20 in blood plasma. These data underscore the critical roles of macrophage-derived IL-10 in limiting atherosclerosis. Supported by grants NIH HL 115232, 145241, and HL088093.

Ectopic expression of an expansin-like B gene from wild Arachis enhances tolerance to both abiotic and biotic stresses.

Plant expansins are structural cell wall-loosening proteins implicated in several developmental processes and responses to environmental constraints and pathogen infection. To date, there is limited information about the biological function of expansins-like B (EXLBs), one of the smallest and less-studied subfamilies of plant expansins. In the present study, we conducted a functional analysis of the wild Arachis AdEXLB8 gene in transgenic tobacco (Nicotiana tabacum) plants to clarify its putative role in mediating defense responses to abiotic and biotic stresses. First, its cell wall localization was confirmed in plants expressing an AdEXLB8:eGFP fusion protein, while nanomechanical assays indicated cell wall reorganization and reassembly due to AdEXLB8 overexpression without compromising the phenotype. We further demonstrated that AdEXLB8 increased tolerance not only to isolated abiotic (drought) and biotic (Sclerotinia sclerotiorum and Meloidogyne incognita) stresses but also to their combination. The jasmonate and abscisic acid signaling pathways were clearly favored in transgenic plants, showing an activated antioxidative defense system. In addition to modifications in the biomechanical properties of the cell wall, we propose that AdEXLB8 overexpression interferes with phytohormone dynamics leading to a defense primed state, which culminates in plant defense responses against isolated and combined abiotic and biotic stresses.

Assigning protein subcellular distributions of vesicle associated tail‐anchored membrane proteins by image‐based machine learning

Subcellular localization of proteins is a key feature of eukaryotes. Traditionally, co-staining with known marker proteins (antibody based or fluorescence fusion protein) or organelle-specific dyes (e.g., MitoTrackerTM) have been used to study subcellular localization of proteins of interest, followed by, a final inspection of the fluorescence microscope images by an experimentalist. However, human visual examination is inclined to bias and membrane proteins are trafficked in vesicles between subcellular locations such that assignment to a specific organelle can be misleading. As an alternative way to assign localization we generated a reference library of confocal micrographs of EGFP fusion proteins localized at key subcellular organelles in murine and human cell lines. Rather than assign the localization of an unknown protein to a specific organelle we consider which reference protein has the most similar subcellular distribution from image sets that are optically validated and comprise 789,011 and 523,319 individual human and murine cell images, respectively. Both morphology and statistical features were computed to enable automated assignment of the subcellular distribution for query proteins by machine learning algorithms with high accuracy. By means of this tool we investigate subcellular transport and distribution for model tail-anchored proteins with randomly mutated C-terminal targeting sequences for the endoplasmic reticulum and through out the secretory pathway.

GhNHX3D, a Vacuolar-Localized Na+/H+ Antiporter, Positively Regulates Salt Response in Upland Cotton

Vacuolar sodium/proton (Na+/H+) antiporters (NHXs) can stabilize ion contents to improve the salt tolerance of plants. Here, GhNHX3D was cloned and characterized from upland cotton (Gossypium hirsutum). Phylogenetic and sequence analyses showed that GhNHX3D belongs to the vacuolar-type NHXs. The GhNHX3D-enhanced green fluorescent protein (eGFP) fusion protein localized on the vacuolar membrane when transiently expressed in Arabidopsis protoplasts. The quantitative real-time PCR (qRT-PCR) analysis showed that GhNHX3D was induced rapidly in response to salt stress in cotton leaves, and its transcript levels increased with the aggravation of salt stress. The introduction of GhNHX3D into the salt-sensitive yeast mutant ATX3 improved its salt tolerance. Furthermore, silencing of GhNHX3D in cotton plants by virus-induced gene silencing (VIGS) increased the Na+ levels in the leaves, stems, and roots and decreased the K+ content in the roots, leading to greater salt sensitivity. Our results indicate that GhNHX3D is a member of the vacuolar NHX family and can confer salt tolerance by adjusting the steady-state balance of cellular Na+ and K+ ions.

Functional analyses of xylanolytic enzymes involved in xylan degradation and utilization in Neurospora crassa

Neurospora crassa possesses six putative xylanases and four putative xylosidases. qRT-PCR results showed that the expression of all these xylanolytic enzymes was induced by xylan. Except for two intracellular β-xylosidases, others were shown to be secreted enzymes based on the localization analysis of EGFP-fusion proteins. Among them, GH10-1, GH10-2, GH11-1, and GH11-2 were successfully expressed and characterized as typical endo-β-1,4-xylanases that hydrolyze the xylooligosaccharides with a polymeric degree not less than three or four. Strains deleted for either gh10-1, gh10-2, gh3-7, or gh3-8 displayed decreased growth in xylan and biomass media. Disruption of gh3-7 or gh43-1 resulted in enhanced-xylanolytic enzyme activity when cultivated in biomass medium. Collectively, these results suggest that xylooligosaccharides released by the actions of xylanases and xylosidases not only serve as the carbon sources to maintain the growth of N. crassa, but they also act as inducers to trigger the expression of hydrolytic enzymes in vivo.

Features of Nuclear Export Signals of NS2 Protein of Influenza D Virus.

Emerging influenza D viruses (IDVs), the newest member in the genus Orthomyxovirus family, which can infect and transmit in multiple mammalian species as its relatives the influenza A viruses (IAVs). Additional studies of biological characteristics of IDVs are needed; here, we studied the characteristics of IDV nonstructural protein 2 (NS2), which shares the lowest homology to known influenza proteins. First, we generated reassortant viruses via reverse genetics to analyze the segment compatibility and gene interchangeability between IAVs and IDVs. Next, we investigated the locations and exact sequences of nuclear export signals (NESs) of the IDV NS2 protein. Surprisingly, three separate NES regions were found to contribute to the nuclear export of an eGFP fusion protein. Alanine scanning mutagenesis identified critical amino acid residues within each NES, and co-immunoprecipitation experiments demonstrated that their nuclear export activities depend on the CRM1-mediated pathway, particularly for the third NES (136-146aa) of IDV NS2. Interestingly, the third NES was important for the interaction of NS2 protein with CRM1. The findings in this study contribute to the understanding of IDV NS2 protein’s role during nucleocytoplasmic transport of influenza viral ribonucleoprotein complexes (vRNPs) and will also facilitate the development of novel anti-influenza drugs targeting nuclear export signals of IDV NS2 protein.

Enhanced X-ray diffraction of in vivo-grown μNS crystals by viscous jets at XFELs.

μNS is a 70 kDa major nonstructural protein of avian reoviruses, which cause significant economic losses in the poultry industry. They replicate inside viral factories in host cells, and the μNS protein has been suggested to be the minimal viral factor required for factory formation. Thus, determining the structure of μNS is of great importance for understanding its role in viral infection. In the study presented here, a fragment consisting of residues 448-605 of μNS was expressed as an EGFP fusion protein in Sf9 insect cells. EGFP-μNS(448-605) crystallization in Sf9 cells was monitored and verified by several imaging techniques. Cells infected with the EGFP-μNS(448-605) baculovirus formed rod-shaped microcrystals (5-15 µm in length) which were reconstituted in high-viscosity media (LCP and agarose) and investigated by serial femtosecond X-ray diffraction using viscous jets at an X-ray free-electron laser (XFEL). The crystals diffracted to 4.5 Å resolution. A total of 4227 diffraction snapshots were successfully indexed into a hexagonal lattice with unit-cell parameters a=109.29, b = 110.29, c = 324.97 Å. The final data set was merged and refined to 7.0 Å resolution. Preliminary electron-density maps were obtained. While more diffraction data are required to solve the structure of μNS(448-605), the current experimental strategy, which couples high-viscosity crystal delivery at an XFEL with in cellulo crystallization, paves the way towards structure determination of the μNS protein.

Boosting carotenoid content in Malus domestica var. Fuji by expressing AtDXR through an Agrobacterium-mediated transformation method.

Apple (Malus domestica) fruits accumulate negligible levels of carotenoids, antioxidant pigments that are precursors for vitamin A in humans. As vitamin A deficiency is an important public health issue, we aimed at increasing carotenoids in apple by constitutively expressing the Arabidopsis thaliana DXR gene, one of the key regulatory steps in the plastidial isoprenoid pathway. For this purpose, we optimized an Agrobacterium-mediated transformation method in the commercial Fuji Raku Raku variety. This resulted in a shoot establishment efficiency of 0.75% at 20 weeks after infection. Molecular and microscopical analyses revealed that 80% of the hygromycin resistant shoots contained and expressed AtDXR:eGFP and that the AtDXR:eGFP fusion protein located in plastids. Transgenic seedlings displayed up to 3-fold increase in total carotenoids and in individual carotenoids compared to the WT, correlating with an increased transcript abundance of endogenous carotenogenic genes such as MdDXS, MdPSY1, MdPSY2, MdPSY3, MdLCYB1, and MdLCYB2. In addition, buds of 2-year-old transgenic dormant trees showed an increment up to 3-fold in lutein, and transient transformation of fruits revealed that AtDXR induced a 2-fold increment in total carotenoids. Thus, these results suggest that DXR may be a good candidate for increasing carotenoid levels in apple fruits through metabolic engineering.

OASTL-A1 functions as a cytosolic cysteine synthase and affects arsenic tolerance in rice.

Arsenic (As) contamination in paddy soil can cause phytotoxicity and elevated As accumulation in rice grains. Arsenic detoxification is closely linked to sulfur assimilation, but the genes involved have not been described in rice. In this study, we characterize the function of OASTL-A1, an O-acetylserine(thiol) lyase, in cysteine biosynthesis and detoxification of As in rice. Tissue expression analysis revealed that OsOASTL-A1 is mainly expressed in roots at the vegetative growth stage and in nodes at the reproductive stage. Furthermore, the expression of OsOASTL-A1 in roots was strongly induced by As exposure. Transgenic rice plants expressing pOsOASTL-A1::GUS (β-glucuronidase) indicated that OsOASTL-A1 was strongly expressed in the outer cortex and the vascular cylinder in the root mature zone. Subcellular localization using OsOASTL-A1:eGFP (enhanced green fluorescent protein) fusion protein showed that OsOASTL-A1 was localized to the cytosol. In vivo and in vitro enzyme activity assays showed that OsOASTL-A1 possessed the O-acetylserine(thiol) lyase activity. Knockout of OsOASTL-A1 led to significantly lower levels of cysteine, glutathione, and phytochelatins in roots and increased sensitivity to arsenate stress. Furthermore, the osoastl-a1 knockout mutants reduced As accumulation in the roots, but increased As accumulation in shoots. We conclude that OsOASTL-A1 is the cytosolic O-acetylserine(thiol) lyase that plays an important role in non-protein thiol biosynthesis in roots for As detoxification.

A reference library for assigning protein subcellular localizations by image-based machine learning.

Confocal micrographs of EGFP fusion proteins localized at key cell organelles in murine and human cells were acquired for use as subcellular localization landmarks. For each of the respective 789,011 and 523,319 optically validated cell images, morphology and statistical features were measured. Machine learning algorithms using these features permit automated assignment of the localization of other proteins and dyes in both cell types with very high accuracy. Automated assignment of subcellular localizations for model tail-anchored proteins with randomly mutated C-terminal targeting sequences allowed the discovery of motifs responsible for targeting to mitochondria, endoplasmic reticulum, and the late secretory pathway. Analysis of directed mutants enabled refinement of these motifs and characterization of protein distributions in within cellular subcompartments.

Recombinant Lactobacillus casei Expressing Capsid Protein VP60 can Serve as Vaccine Against Rabbit Hemorrhagic Disease Virus in Rabbits.

Rabbit hemorrhagic disease virus (RHDV) is the causative agent of rabbit hemorrhagic disease (RHD). RHD, characterized by hemorrhaging, liver necrosis, and high morbidity and mortality in rabbits and hares, causes severe economic losses in the rabbit industry worldwide. Due to the lack of an efficient in-vitro propagation system for RHDV, the current vaccine is produced via chemical inactivation of crude RHDV preparation derived from the livers of infected rabbits. Inactivated vaccines are effective for controlling RHD, but the potential problems of biosafety and animal welfare have negative effects on the application of inactivated vaccines. In this study, an oral Lactobacillus casei (L. casei) vaccine was used as an antigen delivery system to express RHDV capsid protein VP60(VP1)-eGFP fusion protein. The expression of the recombinant protein was confirmed via western blotting and immunofluorescence (IFA). Our results indicate that oral administration of this probiotic vaccine can stimulate secretory immunoglobulin A (SIgA)-based mucosal and IgG-based humoral immune responses in rabbits. The immunized rabbits were completely protected against challenge with RHDV. Our findings indicate that the L. casei expression system is a new strategy for the development of a safe and efficient vaccine against RHDV.