Endogenous Promoter Research Articles

FOXO3 is a latent tumor suppressor for FOXO3-positive and cytoplasmic-type gastric cancer cells

FOXO3 is a member of the FOXO transcription factors thought to play a tumor-suppressor role in gastrointestinal cancer, while tumor-promoting function of FOXO3 has also been reported. These results suggest a context-dependent function of FOXO3 in tumor development. However, the relationship between the FOXO3 expression pattern and its role in tumorigenesis has not been elucidated. We examined the FOXO3 expression in 65 human primary gastric cancer and patient-derived xenograft tissues by immunohistochemistry and identified three subtypes according to subcellular localization: FOXO3-nuclear accumulated (FOXO3-Nuc), FOXO3-nuclear/cytoplasmic or cytoplasmic distributed (FOXO3-Cyt), and FOXO3-negative. In the FOXO3-Cyt gastric cancer cells, the expression of the constitutive active mutant FOXO3 (Act-ER FOXO3) induced the nuclear accumulation of FOXO3 and significantly suppressed colony formation and proliferation. The inhibition of the PI3K-AKT pathway by inhibitor treatment also suppressed the proliferation of FOXO3-Cyt gastric cancer cells, which was associated with the nuclear accumulation of endogenous FOXO3. Furthermore, the expression of Act-ER FOXO3 by an endogenous promoter significantly suppressed gastric tumorigenesis in Gan mice, a model of gastric cancer. Finally, treatment of FOXO3-Cyt human gastric cancer-derived organoids with an AKT inhibitor significantly suppressed the survival and proliferation. These results indicate that FOXO3 is a latent tumor suppressor for FOXO3-Cyt-type gastric cancer cells and that activation of the PI3K-AKT pathway protects this type of gastric cancer cell from FOXO3-mediated growth suppression via constitutive nuclear export. Thus, the inhibition of the PI3K-AKT pathway and nuclear translocation of endogenous FOXO3 may have therapeutic applications in the treatment of FOXO3-positive and cytoplasmic-type gastric cancer.

The functional GRHL3-filaggrin axis maintains a tumor differentiation potential and influences drug sensitivity

Current therapies for treating heterogeneous cancers such as head and neck squamous cell carcinoma (HNSCC) are non-selective and are administered independent of response biomarkers. Therapy resistance subsequently emerges, resulting in increased cellular proliferation that is associated with loss of differentiation. Whether a cancer cell differentiation potential can dictate therapy responsiveness is still currently unknown. A multi-omic approach integrating whole-genome and whole-transcriptome sequencing with drug sensitivity was employed in a HNSCC mouse model, primary patients’ data, and human cell lines to assess the potential of functional differentiation in predicting therapy response. Interestingly, a subset of HNSCC with effective GRHL3-dependent differentiation was the most sensitive to inhibitors of PI3K/mTOR, c-Myc, and STAT3 signaling. Furthermore, we identified the GRHL3-differentiation target gene Filaggrin (FLG) as a response biomarker and more importantly, stratified HNSCC subsets as treatment resistant based on their FLG mutational profile. The loss of FLG in sensitive HNSCC resulted in a dramatic resistance to targeted therapies while the GRHL3-FLG signature predicted a favorable patient prognosis. This study provides evidence for a functional GRHL3-FLG tumor-specific differentiation axis that regulates targeted therapy response in HNSCC and establishes a rationale for clinical investigation of differentiation-paired targeted therapy in heterogeneous cancers.

New inducible mast cell-deficient mouse model (Mcpt5/Cma1DTR)

Mast cell-deficient mice are helpful for understanding the roles of mast cells in vivo. To date, a dozen mouse models for mast cell deficiency have been reported. However, mice with a specific depletion of all populations of mast cells have not been reported. We generated knock-in mice, termed Mcpt5/Cma1DTR mice, expressing human diphtheria toxin A (DT) receptor under the endogenous promoter of Mcpt5 (also known as Cma1), which encodes mouse mast cell protease-5. Flow cytometry and histological analysis showed that intraperitoneal injection of DT induced almost complete depletion of mast cells in heterozygote Mcpt5/Cma1DTR/+ mice. The deletion rates of mast cells in peritoneal cavity, mesentery, abdominal skin, ear skin, and glandular stomach were 99.9%, 100%, 98.7%, 97.7%, and 100%, respectively. Passive cutaneous anaphylaxis reaction also revealed mast cell deficiency in ear skin after DT treatment. Other than mast cells, a small percentage of marginal zone B cells in Mcpt5/Cma1DTR/+ mice were killed by DT treatment. In conclusion, the Mcpt5/Cma1DTR/+ mouse model is valuable for achieving conditional depletion of all populations of mast cells without inducing a marked reduction in other cells.

CRISPR/Cas9-mediated genome editing in Hevea brasiliensis

Clustered regularly Interspaced Short Palindromic Repeats/CRISPR associated protein 9 (CRISPR/Cas9) has not been completely established in Hevea brasiliensis. In the present study, firstly, five endogenous U6 promoters from H. brasiliensis were identified and exploited to drive single guide RNA (sgRNA) transcription for establishing CRISPR/Cas9 transient editing system in H. brasiliensis protoplast. All five promoters were functional, however, their corresponding editing efficiencies were different and varied from 8.47 % to 24.92 %. Secondly, mutation profiles were characterized using 10 sgRNAs targeted five flowering time related genes (HbFT1, HbFT2 and HbTFL1−1, HbTFL1−2, HbTFL1−3) in H. brasiliensis protoplast. Three mutation patterns i. e. deletion, insertion and base substitution were detected. Among these mutations, deletion was the most prominent one and the insertions were observed only in half of 10 target sites, in which the highest insertion frequency (15 %) occurred at TS6 target site. Base substitutions including transition and transversion were detected in 8 out of 10 target sites by deep sequencing. Lastly, stable transformation editing vector targeting phytoene desaturase gene (HbPDS) was constructed and transformed into rubber tree callus, and the “+1” bp homozygous insertions were detected for 3 out of 16 calli at the target site, which turned to expected albino phenotype in the following subculture. This study pronounced the establishment of genome editing in H. brasiliensis by CRISPR/Cas9 plasmid system.

Kozak Sequence Acts as a Negative Regulator for De Novo Transcription Initiation of Newborn Coding Sequences in the Plant Genome.

The manner in which newborn coding sequences and their transcriptional competency emerge during the process of gene evolution remains unclear. Here, we experimentally simulated eukaryotic gene origination processes by mimicking horizontal gene transfer events in the plant genome. We mapped the precise position of the transcription start sites (TSSs) of hundreds of newly introduced promoterless firefly luciferase (LUC) coding sequences in the genome of Arabidopsis thaliana cultured cells. The systematic characterization of the LUC-TSSs revealed that 80% of them occurred under the influence of endogenous promoters, while the remainder underwent de novo activation in the intergenic regions, starting from pyrimidine-purine dinucleotides. These de novo TSSs obeyed unexpected rules; they predominantly occurred ∼100 bp upstream of the LUC inserts and did not overlap with Kozak-containing putative open reading frames (ORFs). These features were the output of the immediate responses to the sequence insertions, rather than a bias in the screening of the LUC gene function. Regarding the wild-type genic TSSs, they appeared to have evolved to lack any ORFs in their vicinities. Therefore, the repulsion by the de novo TSSs of Kozak-containing ORFs described above might be the first selection gate for the occurrence and evolution of TSSs in the plant genome. Based on these results, we characterized the de novo type of TSS identified in the plant genome and discuss its significance in genome evolution.

Design of synthetic promoters for controlled expression of therapeutic genes in retinal pigment epithelial cells.

Age-related macular degeneration (AMD) associated with dysfunction of retinal pigment epithelial (RPE) cells is the most common cause of untreatable blindness. To advance gene therapy as a viable treatment for AMD there is a need for technologies that enable controlled, RPE-specific expression of therapeutic genes. Here we describe design, construction and testing of compact synthetic promoters with a pre-defined transcriptional activity and RPE cell specificity. Initial comparative informatic analyses of RPE and photoreceptor (PR) cell transcriptomic data identified conserved and overrepresented transcription factor regulatory elements (TFREs, 8-19 bp) specifically associated with transcriptionally active RPE genes. Both RPE-specific TFREs and those derived from the generically active cytomegalovirus-immediate early (CMV-IE)promoter were then screened in vitro to identify sequence elements able to control recombinant gene transcription in model induced pluripotent stem (iPS)-derived and primary human RPE cells. Two libraries of heterotypic synthetic promoters varying in predicted RPE specificity and transcriptional activity were designed de novo using combinations of up to 20 discrete TFREs in series (323-602 bp) and their transcriptional activity in model RPE cells was compared to that of the endogenous BEST1 promoter (661 bp, plus an engineered derivative) and the highly active generic CMV-IE promoter (650 bp). Synthetic promoters with a highpredicted specificity, comprised predominantly of endogenous TFREs exhibited a range of activities up to 8-fold that of the RPE-specific BEST1 gene promoter. Moreover, albeit at a lower predicted specificity, synthetic promoter transcriptional activity in model RPE cells was enhanced beyond that of the CMV-IE promoter when viral elements were utilized in combination with endogenous RPE-specific TFREs, with a reduction in promoter size of 15%. Taken together, while our data reveal an inverse relationship between synthetic promoter activity and cell-type specificity, cell context-specific control of recombinant gene transcriptional activity may be achievable.

Role of TNF-α-Inducing Protein Secreted by Helicobacter pylori as a Tumor Promoter in Gastric Cancer and Emerging Preventive Strategies.

The tumor necrosis factor-α (TNF-α)-inducing protein (tipα) gene family, comprising Helicobacter pylori membrane protein 1 (hp-mp1) and tipα, has been identified as a tumor promoter, contributing to H. pylori carcinogenicity. Tipα is a unique H. pylori protein with no similarity to other pathogenicity factors, CagA, VacA, and urease. American H. pylori strains cause human gastric cancer, whereas African strains cause gastritis. The presence of Tipα in American and Euro-Asian strains suggests its involvement in human gastric cancer development. Tipα secreted from H. pylori stimulates gastric cancer development by inducing TNF-α, an endogenous tumor promoter, through its interaction with nucleolin, a Tipα receptor. This review covers the following topics: tumor-promoting activity of the Tipα family members HP-MP1 and Tipα, the mechanism underlying this activity of Tipα via binding to the cell-surface receptor, nucleolin, the crystal structure of rdel-Tipα and N-terminal truncated rTipα, inhibition of Tipα-associated gastric carcinogenesis by tumor suppressor B-cell translocation gene 2 (BTG2/TIS21), and new strategies to prevent and treat gastric cancer. Thus, Tipα contributes to the carcinogenicity of H. pylori by a mechanism that differs from those of CagA and VacA.

A new strategy to develop pseudorabies virus-based bivalent vaccine with high immunogenicity of porcine circovirus type 2

Herpesvirus based multivalent vaccines have been extensively studied, whereas few of them have been successfully used in clinic and animal husbandry industry due to the low expression of foreign immunogens in herpesvirus. In this study, we developed a new strategy to construct herpesvirus based bivalent vaccine with high-level expression of foreign immunogen, by which the ORF2 gene encoding the major antigen protein Cap of porcine circovirus type 2 (PCV2), was highly expressed in pseudorabies virus (PRV). To obtain the high expression of PCV2 immunogen, tandem repeats of PCV2 ORF2 gene were firstly linked by protein quantitation ratioing (PQR) linker to reach equal expression of each ORF2 gene. Then, the multiple copies of ORF2 gene were respectively inserted into the gE and gG sites of PRV using CRISPR/Cas9 system, in which the expression of ORF2 gene was driven by endogenous strong promoters of PRV. Through this way, the highest yield of Cap protein was achieved in two copies of quadruple ORF2 gene insertion. Finally, in mice and pigs immunized with the bivalent vaccine candidate, we detected high titer of specific antibodies for PRV and neutralized antibodies for PCV2, and observed protective effect of the bivalent vaccine candidate against PRV challenge in immunized pigs, suggesting a potential clinical application of the bivalent vaccine candidate we constructed. Together, our strategy could be extensively applied to the generation of other multivalent vaccines, and will pave the way to construct herpesvirus based multivalent vaccines to effectively reduce the cost of vaccine.

SGK1.1 limits brain damage after status epilepticus through M current-dependent and independent mechanisms

Epilepsy is a neurological condition associated to significant brain damage produced by status epilepticus (SE) including neurodegeneration, gliosis and ectopic neurogenesis. Reduction of these processes constitutes a useful strategy to improve recovery and ameliorate negative outcomes after an initial insult. SGK1.1, the neuronal isoform of the serum and glucocorticoids-regulated kinase 1 (SGK1), has been shown to increase M-current density in neurons, leading to reduced excitability and protection against seizures. For this study, we used 4–5 months old male transgenic C57BL/6 J and FVB/NJ mice expressing near physiological levels of a constitutively active form of the kinase controlled by its endogenous promoter. Here we show that SGK1.1 activation potently reduces levels of neuronal death (assessed using Fluoro-Jade C staining) and reactive glial activation (reported by GFAP and Iba-1 markers) in limbic regions and cortex, 72 h after SE induced by kainate, even in the context of high seizure activity. This neuroprotective effect is not exclusively through M-current activation but is also directly linked to decreased apoptosis levels assessed by TUNEL assays and quantification of Bim and Bcl-xL by western blot of hippocampal protein extracts. Our results demonstrate that this newly described antiapoptotic role of SGK1.1 activation acts synergistically with the regulation of cellular excitability, resulting in a significant reduction of SE-induced brain damage in areas relevant to epileptogenesis.

The chloroplast genetic engineering of a unicellular green alga Chlorella vulgaris with two foreign peptides co-expression

The purpose of this study was to establish a chloroplast transformation system in Chlorella vulgaris for expressing recombinant proteins. The 16S-trnI/trnA-23S in the inverted repeat region of C. vulgaris chloroplast genome was used as the flanking fragments in the homologous recombination vector. Meanwhile, the selectable marker aadA gene was promoted by the endogenous 16S promoter, and two codon-optimized antimicrobial peptide NZ2114 and Piscidin-4 genes linked by endogenous ribosome binding site (RBS) in a polycistron were regulated by the rbcL promoter of C. vulgaris. The plasmid was transformed into the chloroplast of C. vulgaris via microparticle bombardment and the transformants were screened using spectinomycin. After multiple rounds' selection, the homoplastic transformants were identified using polymerase chain reaction (PCR), Southern blotting, and Western blotting. This research provided an efficient chloroplast genetic system for C. vulgaris, and will be benefit for the algal further research and application.

Abstract PS17-31: Investigating the estrogen receptor Y537S mutation in transgenic models of luminal B breast cancer

Abstract Breast cancer is the most prevalent cancer in women and over two-thirds of cases express the estrogen receptor (ER). Significantly, metastatic ER positive breast cancer is the leading cause of breast cancer mortality. Despite the success of endocrine therapy to treat ER-positive breast cancers, resistance to treatment is a major problem and relapse commonly occurs though patients initially respond well to treatment. The ER Y537S mutant represents one of the most common gain-of-function mutations detected in the metastatic biopsies of patients with ER-positive breast cancer. Previous in-vitro analyses have shown that the ER Y537S mutant exhibits enhanced ER transcriptional activity by adopting a ligand-independent agonist confirmation. In our study, we seek to provide a deeper understanding of the role of this mutant by modeling the human ER Y537S mutant (ER Y541S in mice). Our lab has generated a Cre-inducible knock-in mouse model expressing the activating point mutation driven by the endogenous ER promoter, and it has been shown that when expressed ubiquitously the mutated ER leads to dramatic developmental effects. In order to further our observations in the context of endocrine resistant metastatic breast cancer, we characterize mamary tumour growth of the ER Y541S mutant in combination with luminal B models of mammary tumourigenesis. Consistent with the notion that these ER mutations occur seldom in primary tumours, preliminary data shows that tumour onset and tumour outgrowth is unaffected by the activating point mutation. When analysing tumours from both ER mutant and wildtype ER control animals, it was found that tumours with mutant ER display more basal-like cytokeratins at the RNA level via RNA sequencing and at the protein level via immunohistochemistry. This expression pattern is often associated with poorer prognosis in the clinic. Through ongoing analyses, we also seek to better understand our model through tumour regression studies (doxycycline withdrawal), treatment with tamoxifen and blocking the main source of estrogens via ovariotomies. By studying the ER Y537S mutation in models of luminal B breast cancer, we hope to investigate the factors leading to tumour recurrence and provide a deeper understanding of potential escape pathways to therapy. Citation Format: Gabriella Johnson, Joshua Roccamo, Cynthia Lavoie, Dongmei Zuo, Virginie Sanguin-Gendreau, Adrian Lee, Zheqi Li, Steffi Oesterreich, William Muller. Investigating the estrogen receptor Y537S mutation in transgenic models of luminal B breast cancer [abstract]. In: Proceedings of the 2020 San Antonio Breast Cancer Virtual Symposium; 2020 Dec 8-11; San Antonio, TX. Philadelphia (PA): AACR; Cancer Res 2021;81(4 Suppl):Abstract nr PS17-31.

Rational design and testing of abiotic stress-inducible synthetic promoters from poplar cis-regulatory elements.

SummaryAbiotic stress resistance traits may be especially crucial for sustainable production of bioenergy tree crops. Here, we show the performance of a set of rationally designed osmotic‐related and salt stress‐inducible synthetic promoters for use in hybrid poplar. De novo motif‐detecting algorithms yielded 30 water‐deficit (SD) and 34 salt stress (SS) candidate DNA motifs from relevant poplar transcriptomes. We selected three conserved water‐deficit stress motifs (SD18, SD13 and SD9) found in 16 co‐expressed gene promoters, and we discovered a well‐conserved motif for salt response (SS16). We characterized several native poplar stress‐inducible promoters to enable comparisons with our synthetic promoters. Fifteen synthetic promoters were designed using various SD and SS subdomains, in which heptameric repeats of five‐to‐eight subdomain bases were fused to a common core promoter downstream, which, in turn, drove a green fluorescent protein (GFP) gene for reporter assays. These 15 synthetic promoters were screened by transient expression assays in poplar leaf mesophyll protoplasts and agroinfiltrated Nicotiana benthamiana leaves under osmotic stress conditions. Twelve synthetic promoters were induced in transient expression assays with a GFP readout. Of these, five promoters (SD18‐1, SD9‐2, SS16‐1, SS16‐2 and SS16‐3) endowed higher inducibility under osmotic stress conditions than native promoters. These five synthetic promoters were stably transformed into Arabidopsis thaliana to study inducibility in whole plants. Herein, SD18‐1 and SD9‐2 were induced by water‐deficit stress, whereas SS16‐1, SS16‐2 and SS16‐3 were induced by salt stress. The synthetic biology design pipeline resulted in five synthetic promoters that outperformed endogenous promoters in transgenic plants.

The ESR1 Mutations: From Bedside to Bench to Bedside.

The ESR1 ligand-binding mutations were unveiled a number of years ago and are the most common genetic mechanism of acquired resistance to endocrine treatment, particularly, to aromatase inhibitors. The discovery of these mutations was enabled after advancements in sequencing technologies and when metastatic tissue samples were interrogated. The ESR1 ligand-binding domain mutations are activating mutations that lead to constitutive ligand-independent activity, which explains the emergence of these mutations under the selective pressure of aromatase inhibitors. Arnesen and colleagues have generated new models of the ESR1 mutations using CRISPR technology to generate single-cell-derived clones in which the ESR1 ligand-binding mutations were "knocked-in" and expressed under the endogenous promoter of estrogen receptor. The authors have extensively characterized these models and have shed new light on the functional consequences ESR1 mutations.See related article by Arnesen et al., p. 539.

APOL1 risk variants affect podocyte lipid homeostasis and energy production in focal segmental glomerulosclerosis.

Lipotoxicity was recently reported in several forms of kidney disease, including focal segmental glomerulosclerosis (FSGS). Susceptibility to FSGS in African Americans is associated with the presence of genetic variants of the Apolipoprotein L1 gene (APOL1) named G1 and G2. If and how endogenous APOL1 may alter mitochondrial function by the modifying cellular lipid metabolism is unknown. Using transgenic mice expressing the APOL1 variants (G0, G1 or G2) under endogenous promoter, we show that APOL1 risk variant expression in transgenic mice does not impair kidney function at baseline. However, APOL1 G1 expression worsens proteinuria and kidney function in mice characterized by the podocyte inducible expression of nuclear factor of activated T-cells (NFAT), which we have found to cause FSGS. APOL1 G1 expression in this FSGS-model also results in increased triglyceride and cholesterol ester contents in kidney cortices, where lipid accumulation correlated with loss of renal function. In vitro, we show that the expression of endogenous APOL1 G1/G2 in human urinary podocytes is associated with increased cellular triglyceride content and is accompanied by mitochondrial dysfunction in the presence of compensatory oxidative phosphorylation (OXPHOS) complexes elevation. Our findings indicate that APOL1 risk variant expression increases the susceptibility to lipid-dependent podocyte injury, ultimately leading to mitochondrial dysfunction.

Single-cell transcriptomic reveals molecular diversity and developmental heterogeneity of human stem cell-derived oligodendrocyte lineage cells

Injury and loss of oligodendrocytes can cause demyelinating diseases such as multiple sclerosis. To improve our understanding of human oligodendrocyte development, which could facilitate development of remyelination-based treatment strategies, here we describe time-course single-cell-transcriptomic analysis of developing human stem cell-derived oligodendrocyte-lineage-cells (hOLLCs). The study includes hOLLCs derived from both genome engineered embryonic stem cell (ESC) reporter cells containing an Identification-and-Purification tag driven by the endogenous PDGFRα promoter and from unmodified induced pluripotent (iPS) cells. Our analysis uncovers substantial transcriptional heterogeneity of PDGFRα-lineage hOLLCs. We discover sub-populations of human oligodendrocyte progenitor cells (hOPCs) including a potential cytokine-responsive hOPC subset, and identify candidate regulatory genes/networks that define the identity of these sub-populations. Pseudotime trajectory analysis defines developmental pathways of oligodendrocytes vs astrocytes from PDGFRα-expressing hOPCs and predicts differentially expressed genes between the two lineages. In addition, pathway enrichment analysis followed by pharmacological intervention of these pathways confirm that mTOR and cholesterol biosynthesis signaling pathways are involved in maturation of oligodendrocytes from hOPCs.

Targeted Gene Delivery: Where to Land.

Genome-editing technologies have the potential to correct most genetic defects involved in blood disorders. In contrast to mutation-specific editing, targeted gene insertion can correct most of the mutations affecting the same gene with a single therapeutic strategy (gene replacement) or provide novel functions to edited cells (gene addition). Targeting a selected genomic harbor can reduce insertional mutagenesis risk, while enabling the exploitation of endogenous promoters, or selected chromatin contexts, to achieve specific transgene expression levels/patterns and the modulation of disease-modifier genes. In this review, we will discuss targeted gene insertion and the advantages and limitations of different genomic harbors currently under investigation for various gene therapy applications.

Differential Effects of Homologous Transcriptional Regulators NicR2A, NicR2B1, and NicR2B2 and Endogenous Ectopic Strong Promoters on Nicotine Metabolism in Pseudomonas sp. Strain JY-Q.

Nicotine is a toxic environmental pollutant that widely exists in tobacco wastes. As a natural nicotine-degrading strain, Pseudomonas sp. strain JY-Q still has difficulties degrading high concentrations of nicotine. In this study, we investigated the effect of two homologous transcriptional regulators and endogenous ectopic strong promoters on the efficiency of nicotine degradation. Comparative genomics analysis showed that two homologous transcriptional regulators, namely, NicR2A and NicR2Bs (NicR2B1 plus NicR2B2), can repress nicotine degradation gene expression. When both nicR2A and nicR2Bs were deleted, the resulting mutant JY-Q ΔnicR2A ΔnicR2B1 ΔnicR2B2 (QΔABs) exhibits a 17% higher nicotine degradation efficiency than wild-type JY-Q. Transcriptome sequencing (RNA-seq) analysis showed that the transcription levels (fragments per kilobase per million [FPKM] value) of six genes were higher than those of the other genes in JY-Q. Based on the genetic organization of these genes, three putative promoters, PRS28250 , PRS09985 , and PRS24685 , were identified. Their promoter activities were evaluated by comparing their expression levels using reverse transcriptase quantitative PCR (RT-qPCR). We found that the transcription levels of RS28250, RS09985, and RS24685 were respectively 16.8, 2.6, and 1.6 times higher than that of hspB2, encoding 6-hydroxy-3-succinylpyridine hydroxylase, which is involved in nicotine degradation. Thus, two strong endogenous promoters, namely, PRS28250 and PRS09985 , were selected to replace the original promoters of nic2 gene clusters. The effect of the endogenous ectopic promoter was also related to the position of target gene clusters. When the promoter PRS28250 replaced the promoter of hspB2, the resultant mutant QΔABs-ΔPhspB2 ::PRS28250 exhibited nicotine-degrading efficiency 69% higher than that of JY-Q. This research suggests a feasible strategy to enhance strains' capacity for nicotine degradation by removal of repressing regulatory proteins and replacing the target promoter with strong endogenous ectopic promoters.IMPORTANCE This study evaluated the differential effects of homologous NicR2A and NicR2Bs and endogenous ectopic strong promoters on nicotine metabolism in Pseudomonas sp. strain JY-Q. Based on our differential analysis, a feasible strategy is presented to modify wild-type (WT) strain JY-Q by removing repressing regulatory proteins NicR2A and NicR2Bs and replacing the target promoter with strong endogenous ectopic promoters. The resulting mutants exhibited high tolerance and degradation of nicotine. These findings should be beneficial for improving the pollutant-degrading capacity of natural strains through genomic modification.

Defining vitamin D receptor expression in the brain using a novel VDRCre mouse.

Vitamin D action has been linked to several diseases regulated by the brain including obesity, diabetes, autism, and Parkinson's. However, the location of the vitamin D receptor (VDR) in the brain is not clear due to conflicting reports. We found that two antibodies previously published as specific in peripheral tissues are not specific in the brain. We thus created a new knockin mouse with cre recombinase expression under the control of the endogenous VDR promoter (VDRCre ). We demonstrated that the cre activity in the VDRCre mouse brain (as reported by a cre-dependent tdTomato expression) is highly overlapping with endogenous VDR mRNAs. These VDR-expressing cells were enriched in multiple brain regions including the cortex, amygdala, caudate putamen, and hypothalamus among others. In the hypothalamus, VDR partially colocalized with vasopressin, oxytocin, estrogen receptor-α, and β-endorphin to various degrees. We further functionally validated our model by demonstrating that the endogenous VDR agonist 1,25-dihydroxyvitamin D activated all tested tdTomato+ neurons in the paraventricular hypothalamus but had no effect on neurons without tdTomato fluorescence. Thus, we have generated a new mouse tool that allows us to visualize VDR-expressing cells and to characterize their functions.

An inducible model for specific neutrophil depletion by diphtheria toxin in mice

Neutrophils are crucial for immunity and play important roles in inflammatory diseases; however, mouse models selectively deficient in neutrophils are limited, and neutrophil-specific diphtheria toxin (DT)-based depletion system has not yet been established. In this study, we generated a novel knock-in mouse model expressing diphtheria toxin receptor (DTR) under control of the endogenous Ly6G promoter. We showed that DTR expression was restricted to Ly6G+ neutrophils and complete depletion of neutrophils could be achieved by DT treatment at 24-48 h intervals. We characterized the effects of specific neutrophil depletion in mice at steady-state, with acute inflammation and during tumor growth. Our study presents a valuable new tool to study the roles of neutrophils in the immune system and during tumor progression.

A FoxL1-CreERT-2A-tdTomato Mouse Labels Subepithelial Telocytes.

A FoxL1-CreERT-2A-tdTomato Mouse Labels Subepithelial Telocytes.