Red Fluorescent Protein Expression Research Articles

Engineering pH-sensitive dissolution of lipid-polymer nanoparticles by Eudragit integration impacts plasmid DNA (pDNA) transfection

Lipid-polymer nanoparticles offer a promising strategy for improving gene nanomedicines by combining the benefits of biocompatibility and stability associated with the individual systems. However, research to date has focused on poly-lactic-co-glycolic acid (PLGA) and resulted in inefficient transfection. In this study, biocompatible Eudragit constructs E100 and RS100 were formulated as lipid-polymer nanoparticles loaded with pDNA expressing red fluorescent protein (RFP) as a model therapeutic. Using a facile nanoprecipitation technique, a core–shell structure stabilised by lipid-polyethylene glycol (PEG) surfactant was produced and displayed resistance to ultracentrifugation. Both cationic polymers E100 (pH-sensitive dissolution at 5) and RS100 (pH-insensitive dissolution) produced 150–200 nm sized particles with a small positive surface charge (+3–5 mV) and high pDNA encapsulation efficiencies (EE) of 75–90%. The dissolution properties of the Eudragit polymers significantly impacted the biological performance in human embryonic kidney cells (HEK293T). Nanoparticles composed of polymer RS100 resulted in consistently high cell viability (80–100%), whereas polymer E100 demonstrated dose-dependent behaviour (20–90% cell viability). The low dissolution of polymer RS100 over the full pH range and the resulting nanoparticles failed to induce RFP expression in HEK293T cells. In contrast, polymer E100-constructed nanoparticles resulted in reproducible and gradually increasing RFP expression of 26–42% at 48–72 h. Intraperitoneal (IP) injection of the polymer E100-based nanoparticles in C57BL/6 mice resulted in targeted RFP expression in mouse testes with favourable biocompatibility one-week post-administration. These findings predicate Eudragit based lipid-polymer nanoparticles as a novel and effective carrier for nucleic acids, which could facilitate pre-clinical evaluation and translation of gene nanomedicines.

Abstract 6930: Spatial analysis of multi-ancestral tumors to understand how intercellular signaling impacts tumor invasion

Abstract Colorectal cancer (CRC) persists as a leading cause of cancer-related deaths in the United States. Intratumoral heterogeneity stems from a multi-ancestral origin or clonal divergence and contributes to treatment resistance. Unfortunately, tumors in existing mouse models are largely homogenous, rendering predictions of drug efficacy inaccurate. To investigate whether signaling between distinct neoplastic clones in a tumor and cells in the surrounding microenvironment impacts CRC biology, we performed spatial transcriptomic analysis on samples collected from a transgenic mouse model previously established in the Halberg laboratory. This model (referred to as B6.FCTG3KM) develops three types of tumors: homotypic Adenomatous Polyposis Coli (APC)-deficient tumors that express red fluorescent protein (RFP), homotypic APC-deficient tumors that express constitutively active phosphoinositide 3-kinase (PI3K) and green fluorescent protein (GFP), and heterotypic tumors. Previous characterization of the model clearly demonstrated that heterotypic tumors are significantly more aggressive than either type of homotypic tumor. Analysis was conducted on four serially sectioned slides containing three tumors and adjacent normal tissue obtained from the colon of a male B6.FCTG3KM mouse using the 10x Genomics Visium CytAssist platform with the mouse whole transcriptome probe set. Collected tumors were staged using traditional histopathology techniques by a board-certified pathologist (Matkowskyj). Immunohistochemistry and immunofluorescence were used to assess β-catenin localization and to re-probe for RFP and GFP expression. Sequencing was performed on the Illumina NovaSeqX platform to an average depth of 255,528,294 reads per sample. Data analysis was performed using the 10x Genomics SpaceRanger and Kendziorski laboratory R/SpatialView packages. A fold-change threshold of 1.5 and an adjusted p-value threshold of 0.05 were used when establishing marker genes for each of the 8 clusters identified. One distinct cluster was identified by marker genes including Notum, Wif1, and Dkk2, factors previously implicated in CRC tumorigenesis, and corresponded to the center of each region of surface-level dysplastic epithelia. The remainder of the surface-level dysplastic epithelia corresponded to a second cluster, with marker genes including those coding for TRIM family proteins (Trim5, Trim10, Trim40, Trim12c), and matrix metalloproteinases (Mmp12, Mmp9, Mmp10, Mmp13). A third cluster corresponded to the desmoplastic stroma underlying each neoplasm. Ongoing analyses are underway to identify clonal interactions that alter gene expression and tumor aggressiveness. Citation Format: Mary M. Stangis, Lingxin Cheng, Dawn Albrecht, Santina Snow, Tessa Wirtz, Kristina A. Matkowskyj, Huy Q. Dinh, Christina Kendziorski, Richard B. Halberg. Spatial analysis of multi-ancestral tumors to understand how intercellular signaling impacts tumor invasion [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2024; Part 1 (Regular Abstracts); 2024 Apr 5-10; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2024;84(6_Suppl):Abstract nr 6930.

Precise control of microfluidic flow conditions is critical for harnessing the in vitro transfection capability of pDNA-loaded lipid-Eudragit nanoparticles.

Microfluidics is widely regarded as a leading technology for industrial-scale manufacture of multicomponent, gene-based nanomedicines in a reproducible manner. Yet, very few investigations detail the impact of flow conditions on the biological performance of the product, particularly biocompatibility and therapeutic efficiency. Herein, this study investigated the engineering of a novel lipid-Eudragit hybrid nanoparticle in a bifurcating microfluidics micromixer for plasmid DNA (pDNA) delivery. Nanoparticles of ~150 nm in size, with uniform polydispersity index (PDI = 0.2) and ξ-potential of 5-11 mV were formed across flow rate ratios (FRR, aqueous to organic phase) of 3:1 and 5:1, respectively. The hybrid nanoparticles maintained colloidal stability and structural integrity of loaded pDNA following recovery by ultracentrifugation. Importantly, in vitro testing in human embryonic kidney cell line (HEK293T) revealed significant differences in biocompatibility and transfection efficiency (TE). Lipid-Eudragit nanoparticles produced at FRR 3:1 displayed high cellular toxicity (0-30% viability), compared with nanoparticles prepared at FRR 5:1 (50-100% viability). Red fluorescent protein (RFP) expression was sustained for 24-72 h following exposure of cells to nanoparticles, indicating controlled release of pDNA and trafficking to the nucleus. Nanoparticles produced at FRR 5:1 resulted in markedly higher TE (12%) compared with those prepared at FRR 3:1 (2%). Notably, nanoparticles produced using the bench-scale nanoprecipitation method resulted in lower biocompatibility (30-90%) but higher RFP expression (25-38%). These findings emphasize the need for in-depth analysis of the effect of formulation and flow conditions on the physicochemical and biological performance of gene nanomedicines when transitioning from bench to clinic.

The Probiotic Bacillus hwajinpoensis Colonizes the Digestive System of Crassostrea gigas Larvae and Protects Them from Vibrio alginolyticus Infection.

The Pacific oyster Crassostrea gigas is one of the most important cultured marine species around the world. Production of Pacific oysters in China has depended primarily on hatchery produced seeds since 2016, with the successful introduction and development of triploid oysters. However, the seed supply of Pacific oysters is threatened by recurring mass mortality events in recent years. Vibriosis is the most commonly encountered disease associated with intensive oyster culture in hatcheries and nurseries. Vibrio alginolyticus and Bacillus hwajinpoensis were the two strains with pathogenic and probiotic effects, respectively, identified during the Pacific oyster larvae production. To monitor their colonization process in Pacific oyster larvae, green fluorescent protein (GFP) and red fluorescent protein (RFP) were labeled to the pathogenic V. alginolyticus and the probiotic B. hwajinpoensis stain, respectively. The pathogenic and probiotic effects of the two strains during the colonization process were then assessed. Stabile expression of GFP and RFP were observed in corresponding stains, and the capabilities of growth, biofilm formation and in vitro adhesion of GFP- and RFP- tagged stains were not significantly different from those of the wild-type strains. Usage of probiotics of 105 CFU/mL significantly inhibited the growth of pathogenic V. alginolyticus and reduced the mortality of D-sharped larvae. Both the pathogenic and probiotic strains employed a similar route to enter and colonize the oyster larvae, which indicates that competing with pathogens for binding and spreading sites were one of the mechanisms of B. hwajinpoensis to provide the probiotic effects to oyster larvae. In summary, employment of fluorescence-tagged pathogenic and probiotic strains simultaneously provides us with an excellent bioassay model to investigate the potential mechanisms of probiotics.

Improved production of β-carotene in light-powered Escherichia coli by co-expression of Gloeobacter rhodopsin expression

BackgroundProviding sufficient and usable energy for the cell factory has long been a heated issue in biosynthesis as solar energy has never been rooted out from the strategy for improvement, and turning Escherichia coli (E. coli) into a phototrophic host has multiple captivating qualities for biosynthesis. In this study, β-carotene was a stable compound for production in E. coli with the expression of four enzymes (CrtE, CrtB, CrtI, CrtY) for production due to its light-harvesting feature as an antenna pigment and as an antioxidant and important precursor for human health. The expression of Gloeobacter rhodopsin (GR) in microbial organisms was proved to have potential for application.ResultsThe expression of fusion protein, GR-GFP, in E. coli showed visible GFP signal under fluorescent microscopy, and its in vivo proton pumping activity signal can be detected in induced photocurrent by electrodes on the chip under intervals of illumination. To assess the phototrophic synthesis ability of the host strain compared to wild-type and vector control strain in chemostat batch with illumination, the expression of red fluorescent protein (RFP) as a target protein showed its yield improvement in protein assay and also reflected its early dominance in RFP fluorescence signal during the incubation, whereas the synthesis of β-carotene also showed yield increase by 1.36-fold and 2.32-fold compared with its wildtype and vector control strain. To investigate the effect of GR-GFP on E. coli, the growth of the host showed early rise into the exponential phase compared to the vector control strain and glucose turnover rate was elevated in increased glucose intake rate and upregulation of ATP-related genes in glycolysis (PtsG, Pgk, Pyk).ConclusionWe reported the first-time potential application of GR in the form of fusion protein GR-GFP. Expression of GR-GFP in E. coli improved the production of β-carotene and RFP. Our work provides a strain of E. coli harboring phototrophic metabolism, thus paving path to a more sustainable and scalable production of biosynthesis.

PTEN knockout using retrogradely transported AAVs transiently restores locomotor abilities in both acute and chronic spinal cord injury

Restoring function in chronic stages of spinal cord injury (SCI) has often been met with failure or reduced efficacy when regenerative strategies are delayed past the acute or sub-acute stages of injury. Restoring function in the chronically injured spinal cord remains a critical challenge. We found that a single injection of retrogradely transported adeno-associated viruses (AAVrg) to knockout the phosphatase and tensin homolog protein (PTEN) in chronic SCI can effectively target both damaged and spared axons and transiently restore locomotor functions in near-complete injury models. AAVrg's were injected to deliver cre recombinase and/or a red fluorescent protein (RFP) under the human Synapsin 1 promoter (hSyn1) into the spinal cords of C57BL/6 PTENFloxΔ/Δ mice to knockout PTEN (PTEN-KO) in a severe thoracic SCI crush model at both acute and chronic time points. PTEN-KO improved locomotor abilities in both acute and chronic SCI conditions over a 9-week period. Regardless of whether treatment was initiated at the time of injury (acute), or three months after SCI (chronic), mice with limited hindlimb joint movement gained hindlimb weight support after treatment. Interestingly, functional improvements were not sustained beyond 9 weeks coincident with a loss of RFP reporter-gene expression and a near-complete loss of treatment-associated functional recovery by 6 months post-treatment. Treatment effects were also specific to severely injured mice; animals with weight support at the time of treatment lost function over a 6-month period. Retrograde tracing with Fluorogold revealed viable neurons throughout the motor cortex despite a loss of RFP expression at 9 weeks post-PTEN-KO. However, few Fluorogold labeled neurons were detected within the motor cortex at 6 months post-treatment. BDA labeling from the motor cortex revealed a dense corticospinal tract (CST) bundle in all groups except chronically treated PTEN-KO mice, indicating a potential long-term toxic effect of PTEN-KO to neurons in the motor cortex which was corroborated by a loss of β-tubulin III labeling above the lesion within spinal cords after PTEN-KO. PTEN-KO mice had significantly more β-tubulin III labeled axons within the lesion when treatment was delivered acutely, but not chronically post-SCI. In conclusion, we have found that using AAVrg's to knockout PTEN is an effective manipulation capable of restoring motor functions in chronic SCI and can enhance axon growth of currently unidentified axon populations when delivered acutely after injury. However, the long-term consequences of PTEN-KO on neuronal health and viability should be further explored.

The pGinger Family of Expression Plasmids.

The pGinger suite of expression plasmids comprises 43 plasmids that will enable precise constitutive and inducible gene expression in a wide range of Gram-negative bacterial species. Constitutive vectors are composed of 16 synthetic constitutive promoters upstream of red fluorescent protein (RFP), with a broad-host-range BBR1 origin and a kanamycin resistance marker. The family also has seven inducible systems (Jungle Express, Psal/NahR, Pm/XylS, Prha/RhaS, LacO1/LacI, LacUV5/LacI, and Ptet/TetR) controlling RFP expression on BBR1/kanamycin plasmid backbones. For four of these inducible systems (Jungle Express, Psal/NahR, LacO1/LacI, and Ptet/TetR), we created variants that utilize the RK2 origin and spectinomycin or gentamicin selection. Relevant RFP expression and growth data have been collected in the model bacterium Escherichia coli as well as Pseudomonas putida. All pGinger vectors are available via the Joint BioEnergy Institute (JBEI) Public Registry. IMPORTANCE Metabolic engineering and synthetic biology are predicated on the precise control of gene expression. As synthetic biology expands beyond model organisms, more tools will be required that function robustly in a wide range of bacterial hosts. The pGinger family of plasmids constitutes 43 plasmids that will enable both constitutive and inducible gene expression in a wide range of nonmodel Proteobacteria.

Tn5 Transposon-based Mutagenesis for Engineering Phage-resistant Strains ofEscherichia coliBL21 (DE3).

Escherichia coli is a preferred strain for recombinant protein production, however, it is often plagued by phage infection during experimental studies and industrial fermentation. While the existing methods of obtaining phage-resistant strains by natural mutation are not efficient enough and time-consuming. Herein, a high-throughput method by combining Tn5 transposon mutation and phage screening was used to produce Escherichia coli BL21 (DE3) phage-resistant strains. Mutant strains PR281-7, PR338-8, PR339-3, PR340-8, and PR347-9 were obtained, and they could effectively resist phage infection. Meanwhile, they had good growth ability, did not contain pseudolysogenic strains, and were controllable. The resultant phage-resistant strains maintained the capabilities of producing recombinant proteins since no difference in mCherry red fluorescent protein expression was found in phage-resistant strains. Comparative genomics showed that PR281-7, PR338-8, PR339-3, and PR340-8 mutated in ecpE, nohD, nrdR, and livM genes, respectively. In this work, a strategy was successfully developed to obtain phage-resistant strains with excellent protein expression characteristics by Tn5 transposon mutation. This study provides a new reference to solve the phage contamination problem.

Efferent network of Neuromedin B neurons in the retrotrapezoid nucleus in mice

In the parafacial region of the medulla, the peptide Neuromedin B (NMB) is expressed in retrotrapezoid nucleus (RTN) neurons that are proposed to be central respiratory chemoreceptors. The efferent network of RTN Nmb neurons has been established in rats, but has not been examined in mice in detail. To map the downstream connections of RTN Nmb neurons we performed genetically-targeted tracing using novel Nmb-Cre knock-in mice. Adult Nmb-Cre mice were injected in the RTN unilaterally with a Cre-dependent AAV producing expression of red fluorescent protein (AAV-Cre ON -mScarlet). Expression of Nmb was identified by fluorescent in situ hybridization (FISH). Injections of AAV-Cre ON -mScarlet in the RTN (N=3) transduced 58 ± 11 % of Nmb-expressing neurons with a selectivity for Nmb neurons of 98 ± 1 %. Intensely labelled axons and terminals were observed in regions overlapping with the medullary and pontine respiratory network. We observe an intra-RTN network of fibers with close appositions between RTN Nmb neurons, as well as moderate projections to areas dorso-medial to the facial nucleus and medially adjacent to the locus coeruleus. There was a modest contralateral projection to the medullary respiratory network, but none to the pontine respiratory network. No labelled fibers were found rostral of the parabrachial nucleus. Anterograde tracing of RTN Nmb neurons was complemented using retrograde transport of Cre-recombinase (AAVrg-Cre) in R26-loxSTOPlox-L10-GFP mice (eGFP-reporter) mice combined with labelling of transcript for Nmb by FISH. Small volumes (3-6 nL) of AAVrg-Cre were unilaterally injected in the Pre-Bötzinger complex (PreBöt), lateral parabrachial nucleus (LPB), caudal ventral respiratory group (cVRG), and the nucleus of the solitary tract (NTS). We determined the proportion of total RTN Nmb neurons that were retro-labelled from each target (PreBöt- 75 ± 3%, n=3; LPB- 65 ± 3%, n=4; cVRG- 54 ± 6%, n=3; NTS- 38 ± 3%, n=3). This result suggests that RTN neurons have a high degree of collateralization within the medullary and pontine respiratory network. Injections of AAVrg-Cre in the PreBöt labelled 16 ± 11% of RTN Nmb neurons contralateral to the injection site. Collectively, these neuroanatomical observations show that the efferent network of RTN Nmb neurons broadly innervate and selectively the central respiratory network in alignment with prior descriptions in rat. Furthermore, we provide evidence for novel connectivity between RTN Nmb neurons. This anatomy supports the idea that RTN neurons generate a coordinated widespread excitation of the respiratory rhythm and pattern generating network in order to promote alveolar ventilation. Funding from NIH-HLBI #HL148004 This is the full abstract presented at the American Physiology Summit 2023 meeting and is only available in HTML format. There are no additional versions or additional content available for this abstract. Physiology was not involved in the peer review process.

Simultaneous expression of three reporter proteins from a porcine reproductive and respiratory syndrome virus-based vector

The mechanism of discontinuous transcription for the synthesis of a series of sub-genomic mRNAs to express the structural proteins of porcine reproductive and respiratory syndrome virus (PRRSV) potentially allows for the simultaneous expression of multiple foreign genes. This can occur by insertion of multiple novel independent transcription units between the ORF sequences of the PRRSV genome. Here, an expression cassette consisting of a red fluorescent protein (RFP) gene flanked at its 3′ end by transcription-regulating sequences (TRS) and an expression cassette consisting of an iLOV gene flanked at its 5′ end by TRS, was constructed. The resulting expression cassette containing a RFP and an iLOV gene were introduced between ORF1b and 2 as well as ORF7 and 3′UTR, respectively, in an infectious PRRSV cDNA clone. Transfection of the resulting clone (pGX-12RFP-73iLOV) into cells resulted in the recovery of a recombinant virus (rGX-12RFP-73iLOV). Simultaneous expression of RFP and iLOV was observed in MARC-145 cells infected with rGX-RFP-iLOV. To test the ability of the PRRSV genome to express all three reporter genes simultaneously, an expression cassette containing the Gluc gene and one containing the iLOV gene were also inserted in between ORF1b and 2 as well as ORF7 and 3′UTR, respectively. This was performed in a recently obtained infectious PRRSV cDNA clone carrying a RFP gene in nsp2. Transfection of the construct (pGX-R-Gluc-iLOV) carrying the three reporter genes into cells allowed the rescue of the recombinant reporter virus (rGX-R-Gluc-iLOV) which showed similar growth characteristics to the parental virus but yielded 100-fold less infectious viruses. Fluorescence microscopy of cells infected with rGX-R-Gluc-iLOV demonstrated the presence of both RFP and iLOV genes. Gluc activities in supernatants harvested at different time points from cells infected with recombinant viruses carrying Gluc showed increased levels of Gluc activity as the infection progressed. This indicated that Gluc gene as well as its activity were acceptable parameters to monitor viral propagation. Our results indicate that it is possible to introduce at least three foreign proteins simultaneously in a PRRSV-based vector and such studies will prove invaluable in our future understanding of these viruses.

Development of porcine induced pluripotent stem cells with a CD163 reporter system

As main recipient cells for porcine reproductive and respiratory syndrome virus (PRRSV), porcine alveolar macrophage (PAM) are involved in the progress of several highly pathogenic virus infections. However, due to the fact that the PAM cells can only be obtained from primary tissues, research on PAM-based virus-host interactions remains challenging. The improvement of induced pluripotent stem cells (iPSCs) technology provides a new strategy to develop IPSCs-derived PAM cells. Since the CD163 is a macrophage-specific marker and a validated receptor essential for PRRSV infection, generation of stable porcine induced pluripotent stem cells lines containing CD163 reporter system play important roles in the investigation of IPSCs-PAM transition and PAM-based virus-host interaction. Based on the CRISPR/Cas9- mediated gene editing system, we designed a sgRNA targeting CD163 locus and constructed the corresponding donor vectors. To test whether this reporter system has the expected function, the reporter system was introduced into primary PAM cells to detect the expression of RFP. To validate the low effect on stem cell pluripotency, we generated porcine iPSC lines containing CD163 reporter and assessed the pluripotency through multiple assays such as alkaline phosphatase staining, immunofluorescent staining, and EdU staining. The red-fluorescent protein (RFP) expression was detected in CD163-edited PAM cells, suggesting that our reporter system indeed has the ability to reflect the expression of gene CD163. Compared with wild-type (WT) iPSCs, the CD163 reporter-iPSCs display similar pluripotency-associated transcription factors expression. Besides, cells with the reporter system showed consistent cell morphology and proliferation ability as compared to WT iPSCs, indicating that the edited-cells have no effect on stem cell pluripotency. In conclusion, we generated porcine iPSCs that contain a CD163 reporter system. Our results demonstrated that this reporter system was functional and safe. This study provides a platform to investigate the iPS-PAM development and virus-host interaction in PAM cells.

CRISPR/Cas9-mediated knockin of IRES-tdTomato at Ins2 locus reveals no RFP-positive cells in mouse islets

Using the CRISPR/Cas9 genomic editing technology, we constructed a transgenic mouse model to express specific fluorescent protein in pancreatic β cells, which harbor tdTomato exogenous gene downstream of the Ins2 promoter in C57BL/6 J mice. The Ins2-specific single-guide RNA-targeted exon2 was designed for the CRISPR/Cas9 system and Donor vector was constructed at the same time. Then Cas9, sgRNA, and Donor vector were microinjected in vitro into the mouse zygotes that were implanted into pseudo-pregnant mice. We obtained homozygotes through mating heterozygotes, and verified the knockin effect through genotype identification, in vivo imaging, and frozen section. Six F0 mice and stable inherited Ins2-IRES-tdTomato F1 were obtained. Genome sequencing results showed that the knockin group had no change in the Ins2 exon compared with the control group, while only the base sequence of tdTomato was added and no base mutation occurred. However, in vivo imaging and frozen section did not observe the expression of red fluorescent protein (RFP), and the protein expression of knockin gene tdTomato was negative. As a result, the expressions of tdTomato protein and fluorescence intensity were low and the detection threshold was not reached. In the CRISP/Cas9 technique, the exogenous fragment of IRES connection would affect the transcription level of the preceding gene, which in turn would lead to low-level expression of the downstream gene and affect the effect of gene insertion.

Intraplacental injection of AAV9-CMV-iCre results in the widespread transduction of multiple organs in double-reporter mouse embryos.

Adeno-associated virus serotype 9 (AAV9) has become a popular tool for gene transfer because of its ability to cross the blood-brain barrier and efficiently transduce genetic material into a variety of cell types. The study utilized GRR (Green-to-Red Reporter) mouse embryos, in which the expression of iCre results in the disappearance of Green Fluorescent Protein (GFP) expression and the detection of Discosoma sp. Red Fluorescent Protein (DsRed) expression by intraplacental injection. Our results demonstrate that AAV9-CMV-iCre can transduce multiple organs in embryos at developmental stages E9.5-E11.5, including the liver, heart, brain, thymus, and intestine. These findings suggest that intraplacental injection of AAV9-CMV-iCre is a viable method for the widespread transduction of GRR mouse embryos.

Extracellular DEK Activates NRF2 Antioxidant Targets and Drives Expansion of Functional and ROS-Low Human Hematopoietic Stem Cells

Umbilical cord blood (CB) is a promising source of hematopoietic stem cells (HSCs) for hematopoietic cell transplantation (HCT), a life-saving treatment, due to lower incidence of graft versus host disease and its availability. CB HCT is limited by low numbers of HSCs, thus there is a strong need to increase numbers of engraftable HSCs, for instance by expanding functionally competent HSCs ex vivo. We have shown that extracellular DEK protein, an epigenetic regulator that is secreted in response to stress and inflammation, is a novel hematopoietic regulatory chemokine. Treatment with extracellular DEK in growth stimulating media significantly enhances the expansion of engraftable HSCs in mouse bone marrow (BM) and human CB (Capitano, et al. JCI 2019; 129(6):2555-2570). To examine mechanisms driving enhanced expansion of functional HSCs after DEK treatment, we performed RNA-sequencing on CB HSCs, multipotent progenitor cells (MPPs), common myeloid progenitors (CMPs), and granulocyte macrophage progenitors (GMPs) after overnight DEK treatment in expansion media. All cell populations upregulated gene programs related to antioxidant responses upon DEK treatment. Indeed, 7/10 genes upregulated by DEK in all HSC/HPC populations (NMRAL2P, PIR, NQO1, SELENOW, FTL, RIT1, and QPCT) promote antioxidant activity. Overnight DEK treatment significantly reduced mean fluorescence of the activated reactive oxygen species (ROS) indicator CM-H2DCFDA in HSCs, MPPs, CMPs, and GMPs. Thus, DEK may induce antioxidant activity. We next found that all 10 genes commonly upregulated in HSCs/HPCs are either direct transcriptional targets of NRF2 (NMRAL2P, LUCAT1,PIR, NQO1, FTL, and QPCT) or are directly affected by changes in NRF2 activation (SELENOW, EBI3, TNFRSF9, and RIT1). Further, several genes highly upregulated by DEK treatment in HSCs (i.e., CCL2, KYNU, and CXCL8) are NRF2 targets. To examine if DEK activates NRF2 transcriptional activity, we utilized a transgene reporter assay where expression of red fluorescent protein (RFP) is driven by activation of promoter antioxidant response elements (ARE), to which active NRF2 binds. DEK treatment of hematopoietic derived cell lines (mouse MLL-AF9 AML and human HL-60 APL) expressing the transgene significantly increased RFP signal, indicating that DEK could activate NRF2. We further examined whether DEK treatment affects mitochondrial metabolism, a source of ROS and tightly regulated process in hematopoiesis. Seahorse XFe analysis, which measures metabolic flux, showed DEK treatment of MLL-AF9 cells significantly reduced mitochondrial oxygen consumption rate, suggesting a reduced reliance on oxidative phosphorylation as an energy source in DEK treated cells. These data suggest that DEK treatment during expansion of primitive hematopoietic cells drives activation of NRF2 transcription, upregulation of antioxidant response genes, reduction of intracellular ROS levels, and may reduce the mitochondrial metabolic rate. These effects in a sense "mimic” the in vivo niche of HSCs, where they reside in low oxygen tensions, have low intracellular ROS, and exhibit low levels of oxidative phosphorylation. To test this hypothesis, we examined if DEK treatment has similar effects to maintaining HSCs in physiologic oxygen tensions (3%) compared to HSCs exposed to extraphysiologic ambient air (21%), which drives oxidative stress (Mantel, et al. Cell 2015; 161(7):1553-65). Isolating and maintaining mouse BM in physiologic oxygen tensions or treating mice in vivo with recombinant DEK preserved HSC numbers compared to exposing the BM cells to ambient air or treating with vehicle. However, combining in vivo DEK treatment with physiologic isolation of HSCs/HPCs had no additive or synergistic effect, suggesting that the overall effects of DEK treatment and maintaining low oxygen tensions may be similar. Thus, extracellular DEK treatment leads to expansion of functionally competent HSCs and reduction of oxidative stress. These data have important implications for the regulation of HSCs/HPCs in stress conditions, where DEK is most likely to be secreted. Further, DEK may present a novel way to mimic physiologic oxygen conditions for enhancement of functional HSC expansion, and the elucidated mechanisms may be exploited for more efficient targeting of molecular programs critical for functional HSC competency, potentially providing new ways to enhance HCT.

\(\textit{n}\)-alkanol stress-induced cell envelope injury of \(σ^{E}\) promoter in \(\textit{Escherichia coli}\)

To characterize the cellular stress by n-alkanols with different alkyl chain lengths in Escherichia coli, we investigated how n-alkanols damage cell envelope permeability and whether they enhance the promoter activity of the envelope stress response regulator, σE, by using variants of green fluorescent protein (GFP). By using E. coli cells having GFPuv expressing and localizing in the cytoplasm, the inner membrane, and the periplasm, after exposure to n-alkanols, the fluorescent intensity of GFPuv released from cells was examined. Our data showed that at the similar levels of cell death of about 90–97%, ethanol, a short-chain alkanol, at a concentration of 20% damaged the outer membrane more greatly than the inner membrane, whereas a longer-chain alkanol of pentanol at a concentration of 1.125% damaged both of the outer and inner membranes. Then we investigated the envelope stress response to n-alkanols by σE factor by ratiometric analysis of rpoE promoter activity for the downstream GFPuv expression referenced to that of housekeeping sigma 70 (σ70 ) recognizing lacUV5 promoter for red fluorescent protein (RFP) expression. The results indicated that the relative activity of rpoE promoter by pentanol was much greater than that of ethanol. The degree of its sensitization by rpoE deficiency was much more remarkable for cells treated with pentanol than for those with ethanol. The results suggest that the response of the σE plays a significant role in the membrane integrity and survival of E. coli cells treated with n-alkanols depending on the alkyl chain length of the molecule.

Conditional Disruption of the BBSome in POMC Neurons Alter Energy and Glucose Homeostasis

The BBSome, a protein complex composed of 8 Bardet‐Biedl syndrome (BBS) proteins including BBS1 has emerged as critical regulator of energy and glucose homeostasis. Humans and animals bearing disruption of the BBSome through loss of various BBS genes display numerous features including obesity and diabetes, but the underlying mechanisms are not well understood. We previously demonstrated that BBSome deficiency in the anorexigenic proopiomelanocortin (POMC) neurons through deletion of the Bbs1 gene lead to the development of obesity. To determine the effects BBSome deficiency in adulthood, we bred mice expressing an inducible POMCERCre with Bbs1fl/fl mice. tdTomato reporter mice were further breed with these mice to validate Cre recombinase via red tdTomato fluorescence protein expression. To induce Bbs1gene deletion we treated the POMCERCre/Bbs1fl/fl at age of 6 weeks with tamoxifen (75 mg/kg, IP, for 5 days). Importantly, both male and female POMCERCre/Bbs1fl/flmice displayed an obesity phenotype as indicated by the increased (P<0.05) body weight (41.0±1.3 vs 36.0±1.9 g in males and 29.0±1.1 vs 26.3±0.9 g in females at age of 20 weeks) and fat mass (10.8±1.2 vs. 7.8.±1.7g in males and 8.2±1.4 vs. 4.8±0.9 g in females). This was associated with increased (P<0.05) food intake (3.3.±0.3 vs. 2.9±0.2 g in males and 2.8±0.4g vs 2.5±0.2 g in females). These results indicate that hyperphagia contributes to the development of obesity in POMCERCre/Bbs1fl/fl mice. Moreover, using glucose tolerance and insulin tolerance tests we found that both male and female POMCERCre/Bbs1fl/flmice display glucose intolerance and insulin resistance. These findings demonstrate the importance of the BBSome in POMC neurons for the regulation of energy and glucose homeostasis.

Orthogonality of Redesigned tRNA Molecules with Three Stop Codons

Comprehensive SummaryThe ability to expand genetic code in living cells has emerged as a powerful method with diverse applications. Here, we design replacement of the anticodons of E. coli tRNAs that recognize codons for 20 natural amino acids, with three anti‐stop codons, resulting in a total of 60 engineered tRNA constructs. We test these constructs one by one in E. coli, and found that six tRNAsCUA (tyrV, serX, hisR, trpT, glnV and leuX), two tRNAsUCA (trpT and leuX) and one tRNAUUA (tyrV) allowed efficient expression of Red Fluorescence Protein (RFP) with the presence of a corresponding stop codon in frame. Furthermore, we exploit the mutual orthogonality of tRNASerCUA, tRNATrpUCA and tRNATyrUUA with corresponding stop codons and demonstrated that the tRNASerCUA and the tRNATrpUCA can provide dynamic range and low crosstalk. Finally, we show the TAG and TGA can not only be used as an “AND gate” circuit to control the translation of target gene, but also be used to control the translation of a prodeoxyviolacein (PDV) pathway and a reporter in parallel. Overall, this work provides flexible tools for translational control and holds great potential to promote synthetic biology studies.

Improvement of 2,3-butanediol production by dCas9 gene expression system in Saccharomyces cerevisiae

Saccharomyces cerevisiae has been widely used in bioproduction. To produce a target product other than ethanol, ethanol production must be decreased to enhance target production. An ethanol non-producing yeast strain was previously constructed by knocking out pyruvate decarboxylase (PDC) genes in the ethanol synthetic pathway. However, glucose uptake by the ethanol-non-producing yeast strain was significantly decreased. In this study, dead Cas9 (dCas9) was used to reduce ethanol synthesis during 2,3-butanediol production without reduction of glucose. The binding site of guide RNA used to effectively suppress PDC1 promoter-driven red fluorescent protein expression by dCas9 was identified and applied to control PDC1 expression. The production of 2,3-butanediol rather than ethanol was improved in repetitive test tube culture. Additionally, ethanol production was decreased and 2,3-butanediol production was increased in the strain expressing dCas9 targeting the PDC1 promoter in the third round of cultivation, compared with the control strain.

Screening and engineering of high-activity promoter elements through transcriptomics and red fluorescent protein visualization in Rhodobacter sphaeroides

The versatile photosynthetic α-proteobacterium Rhodobacter sphaeroides, has recently been extensively engineered as a novel microbial cell factory (MCF) to produce pharmaceuticals, nutraceuticals, commodity chemicals and even hydrogen. However, there are no well-characterized high-activity promoters to modulate gene transcription during the engineering of R. sphaeroides. In this study, several native promoters from R. sphaeroides JDW-710 (JDW-710), an industrial strain producing high levels of co-enzyme Q10 (Q10) were selected on the basis of transcriptomic analysis. These candidate promoters were then characterized by using gusA as a reporter gene. Two native promoters, Prsp_7571 and Prsp_6124, showed 620% and 800% higher activity, respectively, than the tac promoter, which has previously been used for gene overexpression in R. sphaeroides. In addition, a Prsp_7571-derived synthetic promoter library with strengths ranging from 54% to 3200% of that of the tac promoter, was created on the basis of visualization of red fluorescent protein (RFP) expression in R. sphaeroides. Finally, as a demonstration, the synthetic pathway of Q10 was modulated by the selected promoter T334* in JDW-710; the Q10 yield in shake-flasks increased 28% and the production reached 226 mg/L. These well-characterized promoters should be highly useful in current synthetic biology platforms for refactoring the biosynthetic pathway in R. sphaeroides-derived MCFs.

Construction and Characterization of a Gradient Strength Promoter Library for Fine-Tuned Gene Expression in Bacillus licheniformis.

Bacillus licheniformis DW2 is an important industrial strain for bacitracin production, and it is also used for biochemical production, however, the lack of effective toolkit for precise regulation of gene expression hindered its application seriously. Here, a gradient strength promoter library was constructed based on bacitracin synthetase gene cluster promoter PbacA. First, different PbacA promoter variants were constructed via coupling PbacA with various 5'-UTRs, and expression ranges of 32.6-741.8% were attained among these promoters. Then, three promoters, PUbay (strong), PbacA (middle), and PUndh (weakest), were applied for red fluorescent protein (RFP) and keratinase expression assays, and these promoters were proven to have good universality for different proteins. Second, the promoter of bacitracin synthetase gene cluster was replaced by these three promoters, and bacitraicn titer was enhanced by 14.62% when PUbay was applied, which was decreased by 98.05% under the mediation of PUndh compared with that of the original strain DW2. Third, promoters PUbay, PUyvgO, and PUndh were selected to regulate the expression levels of critical genes that are responsible for pucheriminic acid synthesis, and pucheriminic acid yield was increased by 194.1% via manipulating synthetic and competitive pathways. Finally, promoters PUbay, PbacA, and PUndh were applied for green fluorescent protein (GFP) and RFP expression in Escherichia coli, and consistent effects were attained based on our results. Taken together, a gradient strength promoter library was constructed in this research, which provided an effective toolkit for fine-tuning gene expression and reprogramming metabolite metabolic flux in B. licheniformis.