Green Fluorescent Protein Reporter Gene Research Articles

Transcriptional regulation of the yersiniabactin receptor fyuA gene by the ferric uptake regulator in Klebsiella pneumoniae NTUH-K2044.

The ferric uptake regulator (Fur) is a global regulator that influences the expression of virulence genes in Klebsiella pneumoniae. Bioinformatics analysis suggests Fur may involve in iron acquisition via the identified regulatory box upstream of the yersiniabactin receptor gene fyuA. To observe the impact of the gene fyuA on the virulence of K. pneumoniae, the gene fyuA knockout strain and complementation strain were constructed and then conducted a series of phenotypic experiments including chrome azurol S(CAS) detection, crystal violet staining, and wax moth virulence experiment. To examine the regulatory relationship between Fur and the gene fyuA, green fluorescent protein (GFP) reporter gene fusion assay, real-time quantitative reverse transcription polymerase chain reaction (RT-qPCR), gel migration assay (EMSA), and DNase I footprinting assay were used to clarify the regulatory mechanism of Fur on fyuA. CAS detection revealed that the gene fyuA could affect the generation of iron carriers in K. pneumoniae. Crystal violet staining experiment showed that fyuA could positively influence biofilm formation. Wax moth virulence experiment indicated that the deletion of the fyuA could weaken bacterial virulence. GFP reporter gene fusion experiment and RT-qPCRanalysis revealed that Fur negatively regulated the expression of fyuA in iron-sufficient environment. EMSA experiment demonstrated that Fur could directly bind to the promoter region of fyuA, and DNase I footprinting assay further identified the specific binding site sequences. The study showed that Fur negatively regulated the transcriptional expression of fyuA by binding to upstream of the gene promoter region, and then affected the virulence of K. pneumoniae.

Somatic Embryogenesis and Agrobacterium-Mediated Gene Transfer Procedures in Chilean Temperate Japonica Rice Varieties for Precision Breeding

Rice (Oryza sativa) varieties are generated through breeding programs focused on local requirements. In Chile, the southernmost rice producer, rice productivity relies on the use and generation of temperate japonica germplasms, which need to be adapted to the intensifying effects of climate change. Advanced biotechnological tools can contribute to these breeding programs; new technologies associated with precision breeding, including gene editing, rely on procedures such as regeneration and gene transfer. In this study, the local rice varieties Platino, Cuarzo, Esmeralda, and Zafiro were evaluated for somatic embryogenesis potential using a process that involved the combined use of auxins and cytokinins. An auxin-based (2,4-D) general medium (2N6) allowed for the induction of embryogenic masses in all the genotypes. After induction, masses required culturing either in N6R (kinetin; Platino) or N6RN (BAP, kinetin, IBA, and 2,4-D; Cuarzo, Esmeralda, and Zafiro) to yield whole plants using regeneration medium (N6F, no hormone). The sprouting rates indicated Platino as the most responsive genotype; for this reason, this variety was evaluated for gene transfer. Fifteen-day-old embryo masses were assayed for Agrobacterium-mediated transformation using the bacterial strain EHA105 harboring pFLC-Myb/HPT/GFP, a modified T-DNA vector harboring a geminivirus-derived replicon. The vector included the green fluorescent protein reporter gene, allowing for continuous traceability. Reporter mRNA was produced as early as 3 d after agroinfiltration, and stable expression of the protein was observed along the complete process. These achievements enable further biotechnological steps in these and other genotypes from our breeding program.

Establishment and characterization of a muscle cell line from golden pompano (Trachinotus ovatus) and its application in viral susceptibility

A new cell line, golden pompano muscle (GPM), was developed and characterized from the muscle tissue of the golden pompano in this work. GPM cells were mostly fibroblast-like and proliferated well in Leibovitz's L-15 medium supplemented with fetal bovine serum (FBS) (concentration of 10% to 20%) at 28 °C. The cell line has been stably passed over 80 generations and has been successfully cryopreserved. Molecular characterization of cytochrome oxidase subunit I (COI) confirmed that the GPM cells used in this study were from the golden pompano. Chromosome analysis showed that the diploid chromosome number of GPM cells was 2 N = 48. The enhanced green fluorescent protein (EGFP) reporter gene was successfully expressed in the GPM cell line by liposome transfection technique, and the transfection efficiency of GPM cells was about 60%, which suggests that GPM cells are available for research on gene expression in vitro. Virus susceptibility assay showed that GPM cells were susceptible to red-spotted grouper nervous necrosis virus (RGNNV), evidenced by the occurrence of cytopathic effect (CPE) and the increased viral protein synthesis capacity. Furthermore, viral replication significantly increased the expression levels of interferon (IFN) related signaling molecules and pro-inflammatory cytokines in cells. Therefore, our work developed and characterized a new cell line from golden pompano muscle, which allowed for functional studies of genetic manipulation, viral pathogenesis, and the interactions of host and virus.

Design of a sorbitol-activated nitrogen metabolism-dependent regulatory system for redirection of carbon metabolism flow in Bacillus licheniformis.

Synthetic regulation of metabolic fluxes has emerged as a common strategy to improve the performance of microbial cell factories. The present regulatory toolboxes predominantly rely on the control and manipulation of carbon pathways. Nitrogen is an essential nutrient that plays a vital role in growth and metabolism. However, the availability of broadly applicable tools based on nitrogen pathways for metabolic regulation remains limited. In this work, we present a novel regulatory system that harnesses signals associated with nitrogen metabolism to redirect excess carbon flux in Bacillus licheniformis. By engineering the native transcription factor GlnR and incorporating a sorbitol-responsive element, we achieved a remarkable 99% inhibition of the expression of the green fluorescent protein reporter gene. Leveraging this system, we identified the optimal redirection point for the overflow carbon flux, resulting in a substantial 79.5% reduction in acetoin accumulation and a 2.6-fold increase in acetate production. This work highlight the significance of nitrogen metabolism in synthetic biology and its valuable contribution to metabolic engineering. Furthermore, our work paves the way for multidimensional metabolic regulation in future synthetic biology endeavors.

Application of Ha-CoV-2 Pseudovirus for Rapid Quantification of SARS-CoV-2 Variants and Neutralizing Antibodies.

The coronavirus disease 2019 pandemic (COVID-19) has highlighted the need for rapid assays to accurately measure the infectivity of emerging SARS-CoV-2 variants and the effectiveness of vaccine-induced neutralizing antibodies against viral variants. These assays are essential for pandemic surveillance and validating vaccines and variant-specific boosters. This manuscript demonstrates the application of a novel hybrid alphavirus-SARS-CoV-2 pseudovirus (Ha-CoV-2) for quick quantification of SARS-CoV-2 variant infectivity and vaccine-induced neutralizing antibodies to viral variants. Ha-CoV-2 is a SARS-CoV-2 virus-like particle consisting of viral structural proteins (S, M, N, and E) and a fast-expressing RNA genome derived from an alphavirus, Semliki Forest Virus (SFV). Ha-CoV-2 also contains both green fluorescent protein (GFP) and luciferase reporter genes that allow for quick quantification of viral infectivity. As an example, the infectivity of the SARS-CoV-2 Delta (B.1.617.2) and the Omicron (B.1.1.529) variants are quantified, and their sensitivities to a neutralizing antibody (27VB) are also measured. These examples demonstrate the great potential of Ha-CoV-2 as a robust platform for rapid quantification of SARS-CoV-2 variants and their susceptibility to neutralizing antibodies.

In vitro antioxidant, antibacterial, cytotoxic, and epigenetic screening of crude extract and fractions of the marine sponge Neopetrosia exigua from Mauritius waters

The marine sponge Neopetrosia exigua is known as a goldmine of novel compounds, yet its pharmacological activities remain poorly characterised. Herein, this study investigates the bioactivities of N. exigua collected from Mauritius waters. The crude extract (dichloromethane: methanol), hexane, ethyl acetate and aqueous fractions obtained from N. exigua were subjected to in vitro antioxidant assays. Their antibacterial activities were evaluated using the broth microdilution method to determine the minimum inhibitory concentration (MIC). The cytotoxic and epigenetic activities were further screened using the MTT assay and a cell-based image system that measures de-repression of a silenced Green Fluorescent Protein (GFP) reporter gene, respectively. Higher antioxidant activity was recorded for the ethyl acetate fraction as demonstrated by its significant ferric reducing antioxidant power, radical scavenging, and metal chelating activities relative to control (p < 0.05). The best antibacterial profile was presented by the ethyl acetate fraction against Cutibacterium acnes (MIC: 0.039 mg/ml), Streptococcus mutans (MIC: 0.078 mg/ml) and Mycobacterium smegmatis (MIC: 0.313 mg/ml). Similarly, the fraction displayed significant cytotoxicity against the human liposarcoma SW872 cells with IC50 value of 44.34 ± 2.64 µg/ml and GFP re-activation capacity of 43.79 ± 3.19% (p < 0.05). This work conveys interesting data on the antioxidant, antimicrobial, and anticancer properties of N. exigua. In particular, this study indicates the promising potential of N. exigua as a reservoir of epigenetically active agents that can modulate transcription of silenced genes involved in carcinogenesis. Hence, further investigations to isolate the active constituents is actively warranted.

Re-introduction of a bioactive bacterial endophyte back to its seaweed (Ulva sp.) host, influences the host’s microbiome

Ulva is a commercially important marine macroalga. It hosts both epiphytes and endophytes. The latter are assumed to protect Ulva through secondary metabolites. Previously, we demonstrated bioactive endophytes from macroalgae with great potential to control diseases of aquaculture. In this study, we introduced a bioactive bacterial endophyte back into its original host (Ulva sp.) and demonstrated its survival over time in fresh and freeze-dried Ulva sp. We visualized the endophyte’s location and survival in the seaweed using a Green Fluorescent Protein (GFP) reporter gene. The isolate colonized the intercellular space and survived for at least 5 months in fresh, and 12 months in freeze-dried algae, while maintaining its bioactivity against the aquaculture pathogen Streptococcus iniae. We studied the influence of the endophyte on the bacterial community in the Ulva sp. We found that once introduced, the endophyte significantly changed algal microbiota diversity and abundance. Two of Ulva’s associated bacterial species were quantified over time, suggesting different trends in absolute abundance of these bacteria between treatments. To the best of our knowledge, this is the first report of the successful introduction of an endophytic microorganism into macroalgal tissue. These findings may be useful in applied research for the potential management of aquaculture diseases.

A Novel Recombinant Modified Vaccinia Ankara Virus expressing Interleukin-13 Receptor α2 Antigen for Potential Cancer Immunotherapy.

Genetically altered recombinant poxviruses hold great therapeutic promise in animal models of cancer. Poxviruses can induce effective cell-mediated immune responses against tumor-associated antigens. Preventive and therapeutic vaccination with a DNA vaccine expressing IL-13Rα2 can mediate partial regression of established tumors in vivo, indicating that host immune responses against IL-13Rα2 need further augmentation. The aim of the study is developing a recombinant modified vaccinia Ankara (MVA) expressing IL-13RΑ2 (rMVA-IL13RΑ2) virus and study in vitro infectivity and efficacy against IL-13Rα2 positive cell lines. We constructed a recombinant MVA expressing IL-13Rα2 and a green fluorescent protein (GFP) reporter gene. Purified virus titration by infection of target cells and immunostaining using anti-vaccinia and anti-IL-13Rα2 antibodies was used to confirm the identity and purity of the rMVA-IL13Rα2. Western Blot analysis confirmed the presence of IL-13Rα2 protein (~52 kDa). Flow cytometric analysis of IL-13Rα2 negative T98G glioma cells when infected with rMVA-IL13Rα2 virus demonstrated cell-surface expression of IL-13Rα2, indicating the infectivity of the recombinant virus. Incubation of T98G-IL13α2 cells with varying concentrations (0.1-100 ng/ml) of interleukin-13 fused to truncated Pseudomonas exotoxin (IL13-PE) resulted in depletion of GFP+ fluorescence in T98G-IL13Rα2 cells. IL13-PE (10-1000 ng/ml) at higher concentrations also inhibited the protein synthesis in T98G-IL13Rα2 cells compared to cells infected with the control pLW44-MVA virus. IL13-PE treatment of rMVA-IL13Rα2 infected chicken embryonic fibroblast and DF-1 cell line reduced virus titer compared to untreated cells. rMVA-IL13Rα2 virus can successfully infect mammalian cells to express IL-13Rα2 in a biologically active form on the surface of infected cells. To evaluate the efficacy of rMVA-IL13Rα2, immunization studies are planned in murine tumor models.

Optimization of adeno-associated virus (AAV) gene delivery into human bone marrow stem cells (hBMSCs).

Efficiently delivering nucleic acid into mammalian cells is essential to overexpress genes for assessing gene functions. Human bone marrow stem cells (hBMSCs) are the most studied tissue-derived stem cells. Adeno-associated viruses (AAVs) have been used to deliver DNA into hBMSCs for various purposes. Current literature reported that transduction efficiencies of up to 65% could be achieved by AAV gene delivery into hBMSCs. Further improvement of efficiency is needed and possible. This study tested a selection of AAV serotypes for high-efficient DNA delivery into hBMSCs. hBMSCs from different donors were infected with different serotypes of AAVs containing the enhanced green fluorescence protein (eGFP) reporter gene driven by the CMV promoter. Green fluorescence was monitored in the infected cells at five-day intervals. Cells were collected at designated time points after the infection for reverse-transcription polymerase chain reaction (RT-PCR) and quantitative reverse-transcription polymerase chain reaction (qRT-PCR) to assess eGFP mRNA transcription. The results indicated that the order of transduction efficiency of the AAV serotypes was AAV2 > AAV2.7m8 > AAV6 > AAV6.2 > AAV1 > AAV-DJ. AAV2 could achieve almost 100% transduction at the multiplicity of infection (MOI) greater than 100K. Over 90% of cells could be transduced at 20K to 50K MOI. About 80% transduction was seen at MOIs of 10K and 15K. RT-PCR analysis showed that eGFP mRNA could be detected from day 5 to day 30 post-AAV infection. The differences in the observed transduction efficiencies of the hBMSCs from different patients indicate donor-to-donor variability, and increased eGFP mRNA was generally seen after day 15 post-AAV2 infection. Maximal eGFP transcription was detected on day 30 post-infection. We conclude that AAV2 and AAV2.7m8 at an MOI of 100K or greater can efficiently deliver transgene into hBMSCs with up to near 100% transduction efficiency for sustained expression over one month. However, donor-to-donor variation exists in transduction efficiency and transgene expression, especially at MOIs less than 100K.

Drug screening in zebrafish larvae reveals inflammation-related modulators of secondary damage after spinal cord injury in mice.

Prolonged inflammation after spinal cord injury is detrimental to recovery. To find pharmacological modulators of the inflammation response, we designed a rapid drug screening paradigm in larval zebrafish followed by testing of hit compounds in a mouse spinal cord injury model. Methods: We used reduced il-1β linked green fluorescent protein (GFP) reporter gene expression as a read-out for reduced inflammation in a screen of 1081 compounds in larval zebrafish. Hit drugs were tested in a moderate contusion model in mice for cytokine regulation, and improved tissue preservation and locomotor recovery. Results: Three compounds robustly reduced il-1β expression in zebrafish. Cimetidine, an over-the-counter H2 receptor antagonist, also reduced the number of pro-inflammatory neutrophils and rescued recovery after injury in a zebrafish mutant with prolonged inflammation. Cimetidine action on il-1β expression levels was abolished by somatic mutation of H2 receptor hrh2b, suggesting specific action. In mice, systemic treatment with Cimetidine led to significantly improved recovery of locomotor behavior as compared to controls, accompanied by decreased neuronal tissue loss and a shift towards a pro-regenerative profile of cytokine gene expression. Conclusion: Our screen revealed H2 receptor signaling as a promising target for future therapeutic interventions in spinal cord injury. This work highlights the usefulness of the zebrafish model for rapid screening of drug libraries to identify therapeutics to treat mammalian spinal cord injury.

Oct-4 induces cisplatin resistance and tumor stem cell-like properties in endometrial carcinoma cells

ObjectiveResearch has suggested that tumor-initiating tumor stem cells are derived from normal stem cells and that tumor cells undergo progressive de-differentiation to achieve a stem cell-like state. Tumor stem cells are characterized by high proliferation ability, high plasticity, expression of multi-drug resistance proteins, and the ability to seed new tumors. Octamer-binding transcription factor 4 (Oct-4) and its activation targets are overexpressed in the tumor stem cells of various types of tumors, and this expression is associated with the pathogenesis, development, and poor prognosis of tumors. The primary objective of this study was to test if a stably transfected with Oct-4 gene cell line, RL95-2/Oct-4, has the characteristics of tumor stem cells. Materials and methodsHuman endometrial carcinoma cells (RL95-2) were transfected with a plasmid carrying genes for Oct-4 and green fluorescent protein (GFP). The stably transfected cells, RL95-2/Oct-4, were selected using G418 and observed to express the GFP reporter gene under the control of the Oct-4 promoter. GFP expression levels of RL95-2/Oct-4 cells were measured using flow cytometry. The proliferation potential of cells was determined according to cumulative population doubling and colony-formation efficiency. Gene expression was analyzed using reverse transcription-polymerase chain reaction. ResultsRL95-2/Oct-4 cells not only exhibited increased expression of the three most important stem cell genes, Oct-4, Nanog, and Sox2, but also had increased expression of the endometrial tumor stem cell genes CD133 and ALDH1. Furthermore, enhanced expression of these genes in the RL95-2/Oct-4 cells was associated with higher colony-forming ability and growth rate than in parental RL95-2 cells. We also observed that cisplatin induced less cell death in RL95-2/Oct-4 cells than in RL95-2 cells, indicating that RL95-2/Oct-4 cells were more resistant to chemotherapeutic agents. ConclusionThe study findings contribute to investigate the effects of Oct-4 on tumor stem cell origins.

Establishment and application of four long-term culture cell lines of the olive flounder Paralichthys olivaceus blastocysts

Four new embryonic cell lines derived from blastocysts of the olive flounder Paralichthys olivaceus, an important commercial marine fish, were established and characterized. They were designated as PoEFCI, PoEFCII, PoEFCIII, and PoEFCIV and were all fibroblastic cells. The cells were cultured in DMEM/F-12 medium supplemented with antibiotics, FBS, and growth factors at temperature of 25 °C and subcultured for >100 passages over 18 months. The origin of the cell lines was confirmed by examining the partial sequences of the cytochrome oxidase c subunit I (COI) gene of the flounder mitochondrial DNA (mtDNA). The four cell lines showed different growth curve patterns. According to the results of gene and protein expression and enzyme activity, the cell lines PoEFCI, PoEFCII, and PoEFC III could be pluripotent. The cells of all four cell lines were also successfully transfected with the green fluorescent protein (GFP) reporter gene, suggesting that they could be used to study gene function in the flounder or other fish. More importantly, PoEFCI-III were sensitive to chromium (Cr) and red sea bream Pagrus major iridovirus (RSIV), so they could be used as a powerful tool for the study of the toxicological investigation of heavy metals and RSIV in fish. Therefore, these cell lines would be useful for biotechnological and toxicological research on marine fish as an in vitro biological system.

A new embryonic cell line YCE1 from large yellow croaker (Larimichthys crocea) and its susceptibility to large yellow croaker iridovirus

In order to develop in vitro culture models for the viral pathogens found in large yellow croaker (Larimichthys crocea), a new cell line derived from L. crocea embryo, YCE1, was established and characterized. This cell line has been maintained in vitro for over 24 months, and subcultured for >80 times in a Leibovitz's L-15 medium supplemented with fetal bovine serum (FBS). The YCE1 cells, which were identified to be predominantly fibroblast-like by immunofluorescence, are able to form a monolayer at 15–37 °C, with an optimum growth temperature of 26 °C. In the chromosome analysis, the YCE1 cells exhibited to be highly heteroploid. The green fluorescence protein (GFP) reporter gene was successfully expressed in the YCE1 cells. In the susceptibility test with regard to large yellow croaker iridovirus (LYCIV) in the YCE1 cells, noticeable and stable cytopathic effects (CPEs) were found in a host-origin cell line for the first time. In addition, a special intracellular pattern of the immature viral particles contained in vesicular organelles was confirmed by transmission electron microscopy, suggesting that the YCE1 cell line could serve as a viable tool for the investigation of host-pathogen interaction of LYCIV and other related megalocytiviruses.

DNA-free genome editing in grapevine using CRISPR/Cas9 ribonucleoprotein complexes followed by protoplast regeneration.

CRISPR/Cas9 genome editing technology can overcome many limitations of traditional breeding, offering enormous potential for crop improvement and food production. Although the direct delivery of Cas9-single guide RNA (sgRNA) ribonucleoprotein (RNP) complexes to grapevine (Vitis vinifera) protoplasts has been shown before, the regeneration of edited protoplasts into whole plants has not been reported. Here, we describe an efficient approach to obtain transgene-free edited grapevine plants by the transfection and subsequent regeneration of protoplasts isolated from embryogenic callus. As proof of concept, a single-copy green fluorescent protein reporter gene (GFP) in the grapevine cultivar Thompson Seedless was targeted and knocked out by the direct delivery of RNPs to protoplasts. CRISPR/Cas9 activity, guided by two independent sgRNAs, was confirmed by the loss of GFP fluorescence. The regeneration of GFP- protoplasts into whole plants was monitored throughout development, confirming that the edited grapevine plants were comparable in morphology and growth habit to wild-type controls. We report the first highly efficient protocol for DNA-free genome editing in grapevine by the direct delivery of preassembled Cas9-sgRNA RNP complexes into protoplasts, helping to address the regulatory concerns related to genetically modified plants. This technology could encourage the application of genome editing for the genetic improvement of grapevine and other woody crop plants.

Rescue of the first alphanucleorhabdovirus entirely from cloned complementary DNA: An efficient vector for systemic expression of foreign genes in maize and insect vectors.

Recent reverse genetics technologies have enabled genetic manipulation of plant negative-strand RNA virus (NSR) genomes. Here, we report construction of an infectious clone for the maize-infecting Alphanucleorhabdovirus maydis, the first efficient NSR vector for maize. The full-length infectious clone was established using agrobacterium-mediated delivery of full-length maize mosaic virus (MMV) antigenomic RNA and the viral core proteins (nucleoprotein N, phosphoprotein P, and RNA-directed RNA polymerase L) required for viral transcription and replication into Nicotiana benthamiana. Insertion of intron 2 ST-LS1 into the viral L gene increased stability of the infectious clone in Escherichia coli and Agrobacterium tumefaciens. To monitor virus infection in vivo, a green fluorescent protein (GFP) gene was inserted in between the N and P gene junctions to generate recombinant MMV-GFP. Complementary DNA (cDNA) clones of MMV-wild type (WT) and MMV-GFP replicated in single cells of agroinfiltrated N.benthamiana. Uniform systemic infection and high GFP expression were observed in maize inoculated with extracts of the infiltrated N.benthamiana leaves. Insect vectors supported virus infection when inoculated via feeding on infected maize or microinjection. Both MMV-WT and MMV-GFP were efficiently transmitted to maize by planthopper vectors. The GFP reporter gene was stable in the virus genome and expression remained high over three cycles of transmission in plants and insects. The MMV infectious clone will be a versatile tool for expression of proteins of interest in maize and cross-kingdom studies of virus replication in plant and insect hosts.

Lipid nanoparticle formulations for optimal RNA-based topical delivery to murine airways

Introduction: Lipid nanoparticles (LNP) have been successfully used as a platform technology for delivering nucleic acids to the liver. To broaden the application of LNPs in targeting non-hepatic tissues, we developed LNP-based RNA therapies (siRNA or mRNA) for the respiratory tract. Such optimized LNP systems could offer an early treatment strategy for viral respiratory tract infections such as COVID-19.Methods: We generated a small library of six LNP formulations with varying helper lipid compositions and characterized their hydrodynamic diameter, size distribution and cargo entrapment properties. Next, we screened these LNP formulations for particle uptake and evaluated their potential for transfecting mRNA encoding green fluorescence protein (GFP) or SARS-CoV2 nucleocapsid-GFP fusion reporter gene in a human airway epithelial cell line in vitro. Following LNP-siGFP delivery, GFP protein knockdown efficiency was assessed by flow cytometry to determine %GFP+ cells and median fluorescence intensity (MFI) for GFP. Finally, lead LNP candidates were validated in Friend leukemia virus B (FVB) male mice via intranasal delivery of an mRNA encoding luciferase, using in vivo bioluminescence imaging.Results: Dynamic light scattering revealed that all LNP formulations contained particles with an average diameter of <100 nm and a polydispersity index of <0.2. Human airway epithelial cell lines in culture internalized LNPs with differential GFP transfection efficiencies (73–97%). The lead formulation LNP6 entrapping GFP or Nuc-GFP mRNA demonstrated the highest transfection efficiency (97%). Administration of LNP-GFP siRNA resulted in a significant reduction of GFP protein expression. For in vivo studies, intranasal delivery of LNPs containing helper lipids (DSPC, DOPC, ESM or DOPS) with luciferase mRNA showed significant increase in luminescence expression in nasal cavity and lungs by at least 10 times above baseline control.Conclusion: LNP formulations enable the delivery of RNA payloads into human airway epithelial cells, and in the murine respiratory system; they can be delivered to nasal mucosa and lower respiratory tract via intranasal delivery. The composition of helper lipids in LNPs crucially modulates transfection efficiencies in airway epithelia, highlighting their importance in effective delivery of therapeutic products for airways diseases.

Genetic transformation of clone rootstock of stone fruits 146-2 using the green fluorescent protein reporter gene

For the form of dwarf winter-hardy clonal rootstocks of stone crops146-2 (Prunus pumilaL.xP.tomentosaThunb.), system of regeneration and genetic transformation using green fluorescent protein (GFP) has been developed. For eff ective regeneration of accessory shoots, no pre-treatment with 6-benzylamine-purine (BA) and auxin was required. Stimulation of the regeneration of shoots from leaf explants required 2-3 weeks of a dark period. Th e best percentage of regeneration (greater than 75 %) was observed with a combination of 3 mg/L BA and 0.75 mg/L IBA. The achieved regeneration effi ciency made it possible to develop a protocol for genetic transformation, mediated byAgrobacterium, for rootstock 146-2. Whole leaves from in vitro-cultured shoots were used as explants for transformation by theA. tumefaciensstrain CBE21, with the binary vector pBINmGFP5ER containing thenptIIencoding neomycin phosphotransferase II as a plant-selectable marker under the control of the NOS promoter (nopalin synthase) and the reportergfpgene encoding a green fluorescent protein under the control of the cauliflower mosaic virus (CaMV) promoter 35S. Th e integration ofnptIIandgfpinto transgenes was confirmed by PCR. Expression of the green fluorescent protein was observed using fluorescence microscopy. The efficiency of transformation based on PCR analysis of independent lines resistant to kanamycin was 0.41-0.83 %. All transgenic lines showed resistance to kanamycin at a concentration of 40 mg/L. They were rooted and acclimatized to greenhouse conditions. Th e developed protocols will be used to producePlum pox virus(PPV) resistant plants.

A Unique Robust Dual-Promoter-Driven and Dual-Reporter-Expressing SARS-CoV-2 Replicon: Construction and Characterization.

The severe acute respiratory syndrome coronavirus-2 (SARS-CoV-2, SARS2) remains a great global health threat and demands identification of more effective and SARS2-targeted antiviral drugs, even with successful development of anti-SARS2 vaccines. Viral replicons have proven to be a rapid, safe, and readily scalable platform for high-throughput screening, identification, and evaluation of antiviral drugs against positive-stranded RNA viruses. In the study, we report a unique robust HIV long terminal repeat (LTR)/T7 dual-promoter-driven and dual-reporter firefly luciferase (fLuc) and green fluorescent protein (GFP)-expressing SARS2 replicon. The genomic organization of the replicon was designed with quite a few features that were to ensure the replication fidelity of the replicon, to maximize the expression of the full-length replicon, and to offer the monitoring flexibility of the replicon replication. We showed the success of the construction of the replicon and expression of reporter genes fLuc and GFP and SARS structural N from the replicon DNA or the RNA that was in vitro transcribed from the replicon DNA. We also showed detection of the negative-stranded genomic RNA (gRNA) and subgenomic RNA (sgRNA) intermediates, a hallmark of replication of positive-stranded RNA viruses from the replicon. Lastly, we showed that expression of the reporter genes, N gene, gRNA, and sgRNA from the replicon was sensitive to inhibition by Remdesivir. Taken together, our results support use of the replicon for identification of anti-SARS2 drugs and development of new anti-SARS strategies targeted at the step of virus replication.

Tumor-associated macrophage heterogeneity is driven by tissue territories in breast cancer.

Tissue-resident macrophages adapt to local signals within tissues to acquire specific functions. Neoplasia transforms the tissue, raising the question as to how the environmental perturbations contribute to tumor-associated macrophage (TAM) identity and functions. Combining single-cell RNA sequencing (scRNA-seq) with spatial localization of distinct TAM subsets by imaging, we discover that TAM transcriptomic programs follow two main differentiation paths according to their localization in the stroma or in the neoplastic epithelium of the mammary duct. Furthermore, this diversity is exclusively detected in a spontaneous tumor model and tracks the different tissue territories as well as the type of tumor lesion. These TAM subsets harbor distinct capacity to activate CD8+ Tcells and phagocyte tumor cells, supporting that specific tumor regions, rather than defined activation states, are the major drivers of TAM plasticity and heterogeneity. The distinctions created here provide a framework to design cancer treatment targeting specific TAM niches.

The homologous region hr4a of Autographa californica multiple nucleopolyhedrovirus specifically enhances viral early promoters

Autographa californica multiple nucleocapsid polyhedrosis virus (AcMNPV) genome contains several homologous regions (hrs) which can be used as origins of viral DNA replication and transcriptional enhancers. Early studies have found that some hrs can not only stimulate viral early promoters, but also enhance gene expression driven by viral late promoters. In this study, by adding hr4a to the upstream of different types of viral promoters and enhanced green fluorescent protein (egfp) reporter gene, we investigated the enhancement effect of hr4a on eGFP expression. The results showed that hr4a increased the gene transcription controlled by early promoters, but had no enhancer effect on late promoters.