An expanded genetic toolkit for inducible expression and targeted gene silencing in Rickettsia parkeri.

Pathogenic species within the Rickettsia genus are transmitted to humans through arthropod vectors and cause a spectrum of diseases ranging from mild to life-threatening. Despite rickettsiae posing an emerging global health risk, the genetic requirements of their infectious life cycles remain poorly understood. A major hurdle toward building this understanding has been the lack of efficient tools for genetic manipulation, owing to the technical difficulties associated with their obligate intracellular nature. To this end, we implemented the Tet-On system to enable conditional gene expression in Rickettsia parkeri. Using Tet-On, we show inducible expression of antibiotic resistance and a fluorescent reporter. We further used this inducible promoter to screen the ability of R. parkeri to express four variants of the catalytically dead Cas9 (dCas9). We demonstrate that all four dCas9 variants can be expressed in R. parkeri and used for CRISPR interference (CRISPRi)-mediated targeted gene knockdown. We show targeted knockdown of an antibiotic resistance gene as well as the endogenous virulence factor sca2. Altogether, we have developed systems for inducible gene expression and CRISPRi-mediated gene knockdown for the first time in rickettsiae, laying the groundwork for more scalable, targeted mechanistic investigations into their infectious life cycles.IMPORTANCEThe spotted fever group of Rickettsia contains vector-borne pathogenic bacteria that are neglected and emerging threats to public health. Due to the obligate intracellular nature of rickettsiae, the development of tools for genetic manipulation has been stunted, and the molecular and genetic underpinnings of their infectious lifecycle remain poorly understood. Here, we expand the genetic toolkit by introducing systems for conditional gene expression and CRISPR interference (CRISPRi)-mediated gene knockdown. These systems allow for relatively easy manipulation of rickettsial gene expression. We demonstrate the effectiveness of these tools by disrupting the intracellular life cycle using CRISPRi to deplete the sca2 virulence factor. These tools will be crucial for building a more comprehensive and detailed understanding of rickettsial biology and pathogenesis.

Riboswitch-controlled IL-12 gene therapy reduces hepatocellular cancer in mice.

Hepatocellular carcinoma (HCC) and solid cancers with liver metastases are indications with high unmet medical need. Interleukin-12 (IL-12) is a proinflammatory cytokine with substantial anti-tumor properties, but its therapeutic potential has not been realized due to severe toxicity. Here, we show that orthotopic liver tumors in mice can be treated by targeting hepatocytes via systemic delivery of adeno-associated virus (AAV) vectors carrying the murine IL-12 gene. Controlled cytokine production was achieved in vivo by using the tetracycline-inducible K19 riboswitch. AAV-mediated expression of IL-12 led to STAT4 phosphorylation, interferon-γ (IFNγ) production, infiltration of T cells and, ultimately, tumor regression. By detailed analyses of efficacy and tolerability in healthy and tumor-bearing animals, we could define a safe and efficacious vector dose. As a potential clinical candidate, we characterized vectors carrying the human IL-12 (huIL-12) gene. In mice, bioactive human IL-12 was expressed in a vector dose-dependent manner and could be induced by tetracycline, suggesting tissue-specific AAV vectors with riboswitch-controlled expression of highly potent proinflammatory cytokines as an attractive approach for vector-based cancer immunotherapy.

miR-200c-141 induces a hybrid E/M state and promotes collective cell migration in MDA-MB-231 cells.

The microRNA-200 (miR-200) family is a potent suppressor of epithelial-to-mesenchymal transition (EMT). While its role as a tumor suppressor has been well documented, recent studies suggested that it can promote cancer progression in several stages. In this study, we investigated whether the miR-200 family members play a role in the acquisition of a hybrid epithelial/mesenchymal (E/M) state, which is reported to be associated with cancer malignancy, in mesenchymal MDA-MB-231 cells. Our results demonstrated that the induction of miR-200c-141, a cluster of the miR-200 family member, can induce the expression of epithelial gene and cell-cell junction while mesenchymal markers are retained. Moreover, induction of miR-200c-141 promoted collective migration accompanied by the formation of F-actin cables anchored by adherens junction. These results suggest that the miR-200 family can induce a hybrid E/M state and endows with the ability of collective cell migration in mesenchymal cancer cells.

A novel transgenic mouse line with hippocampus-dominant and inducible expression of truncated human tau.

BACKGROUND: Intraneuronal accumulation of hyperphosphorylated tau is a defining hallmark of Alzheimer's disease (AD). However, mouse models imitating AD-exclusive neuronal tau pathologies are lacking. METHODS: We generated a new tet-on transgenic mouse model expressing truncated human tau N1-368 (termed hTau368), a tau fragment increased in the brains of AD patients and aged mouse brains. Doxycycline (dox) was administered in drinking water to induce hTau368 expression. Immunostaining and Western blotting were performed to measure the tau level. RNA sequencing was performed to evaluate gene expression, and several behavioral tests were conducted to evaluate mouse cognitive functions, emotion and locomotion. RESULTS: Dox treatment for 1-2 months at a young age induced overt and reversible human tau accumulation in the brains of hTau368 transgenic mice, predominantly in the hippocampus. Meanwhile, the transgenic mice exhibited AD-like high level of tau phosphorylation, glial activation, loss of mature neurons, impaired hippocampal neurogenesis, synaptic degeneration and cognitive deficits. CONCLUSIONS: This study developed a well-characterized and easy-to-use tool for the investigations and drug development for AD and other tauopathies.

TOR Signaling Tightly Regulated Vegetative Growth, Conidiation, Oxidative Stress Tolerance and Entomopathogenicity in the Fungus Beauveria bassiana.

Beauveria bassiana degenerates after repeated subcultures, demonstrating declined conidiation and insect virulence. The target of rapamycin (TOR) kinase conserved among eukaryotes is the master regulator of cellular physiology and is likely involved in culture degeneration. Indeed, the levels of TOR-associated proteins increase over successive subcultures. Here, CRISPR/Cas9 locus engineering introduced the inducible Tet-On promoter upstream of the TOR kinase 2 gene tor2 in B. bassiana. The mutant PTet-Ontor2 'T41' was verified for the Tet-On integration via PCR analyses and provided a model for evaluating the fungal phenotypes according to the tor2 expression levels, induced by doxycycline (Dox) concentrations. At 0 µg·mL-1 of Dox, T41 had 68% of the wild type's (WT) tor2 expression level, hampered radial growth and relatively lower levels of oxidative stress tolerance, conidiation and virulence against Spodoptera exigua, compared to those under the presence of Dox. A low dose of Dox at 0.1-1 µg·mL-1 induced tor2 upregulation in T41 by up to 91% compared to 0 µg·mL-1 of Dox, resulting in significant increases in radial growth by 8-10% and conidiation by 8-27%. At 20 µg·mL-1 of Dox, which is 132% higher than T41's tor2 expression level at 0 µg·mL-1 of Dox, T41 showed an increased oxidative stress tolerance and a decrease in growth inhibition under iron replete by 62%, but its conidiation significantly dropped by 47% compared to 0 µg·mL-1 of Dox. T41 at 20 µg·mL-1 of Dox had a strikingly increased virulence (1.2 day lower LT50) against S. exigua. The results reflect the crucial roles of TOR kinase in the vegetative growth, conidiation, pathogenicity and oxidative stress tolerance in B. bassiana. Since TOR upregulation is correlated with culture degeneration in multiple subcultures, our data suggest that TOR signaling at relatively low levels plays an important role in growth and development, but at moderate to high levels could contribute to some degenerated phenotypes, e.g., those found in successive subcultures.

[Establishment of leukemia cell model with inducible AML1-ETO expression and its effect on fatty acid metabolism in leukemia cells].

Objective: To investigate the effect of the AML1-ETO (AE) fusion gene on the biological function of U937 leukemia cells by establishing a leukemia cell model that induces AE fusion gene expression. Methods: The doxycycline (Dox) -dependent expression of the AE fusion gene in the U937 cell line (U937-AE) were established using a lentivirus vector system. The Cell Counting Kit 8 methods, including the PI and sidanilide induction, were used to detect cell proliferation, cell cycle-induced differentiation assays, respectively. The effect of the AE fusion gene on the biological function of U937-AE cells was preliminarily explored using transcriptome sequencing and metabonomic sequencing. Results: ①The Dox-dependent Tet-on regulatory system was successfully constructed to regulate the stable AE fusion gene expression in U937-AE cells. ②Cell proliferation slowed down and the cell proliferation rate with AE expression (3.47±0.07) was lower than AE non-expression (3.86 ± 0.05) after inducing the AE fusion gene expression for 24 h (P<0.05). The proportion of cells in the G(0)/G(1) phase in the cell cycle increased, with AE expression [ (63.45±3.10) %) ] was higher than AE non-expression [ (41.36± 9.56) %] (P<0.05). The proportion of cells expressing CD13 and CD14 decreased with the expression of AE. The AE negative group is significantly higher than the AE positive group (P<0.05). ③The enrichment analysis of the transcriptome sequencing gene set revealed significantly enriched quiescence, nuclear factor kappa-light-chain-enhancer of activated B cells, interferon-α/γ, and other inflammatory response and immune regulation signals after AE expression. ④Disorder of fatty acid metabolism of U937-AE cells occurred under the influence of AE. The concentration of the medium and short-chain fatty acid acylcarnitine metabolites decreased in cells with AE expressing, propionyl L-carnitine, wherein those with AE expression (0.46±0.13) were lower than those with AE non-expression (1.00±0.27) (P<0.05). The metabolite concentration of some long-chain fatty acid acylcarnitine increased in cells with AE expressing tetradecanoyl carnitine, wherein those with AE expression (1.26±0.01) were higher than those with AE non-expression (1.00±0.05) (P<0.05) . Conclusion: This study successfully established a leukemia cell model that can induce AE expression. The AE expression blocked the cell cycle and inhibited cell differentiation. The gene sets related to the inflammatory reactions was significantly enriched in U937-AE cells that express AE, and fatty acid metabolism was disordered.

Gene Augmentation for Autosomal Dominant CRX-Associated Retinopathies.

The cone-rod homeobox (CRX) protein is a key transcription factor essential for photoreceptor function and survival. Mutations in human CRX gene are linked to a wide spectrum of blinding diseases ranging from mild macular dystrophy to severe Leber congenital amaurosis (LCA), cone-rod dystrophy (CRD), and retinitis pigmentosa (RP). These diseases are still incurable and mostly inherited in an autosomal dominant form. Dysfunctional mutant CRX protein interferes with the function of wild-type CRX protein, demonstrating the dominant negative effect. At present, gene augmentation is the most promising treatment strategy for hereditary diseases. This study aims to review the pathogenic mechanisms of various CRX mutations and propose two therapeutic strategies to rescue sick photoreceptors in CRX-associated retinopathies, namely, Tet-On-hCRX system and adeno-associated virus (AAV)-mediated gene augmentation. The outcome of proposed studies will guide future translational research and suggest guidelines for therapy evaluation in terms of treatment safety and efficacy.

Regression modelling of conditional morphogene expression links and quantifies the impact of growth rate, fitness and macromorphology with protein secretion in Aspergillus niger.

BACKGROUND: Filamentous fungi are used as industrial cell factories to produce a diverse portfolio of proteins, organic acids, and secondary metabolites in submerged fermentation. Generating optimized strains for maximum product titres relies on a complex interplay of molecular, cellular, morphological, and macromorphological factors that are not yet fully understood. RESULTS: In this study, we generate six conditional expression mutants in the protein producing ascomycete Aspergillus niger and use them as tools to reverse engineer factors which impact total secreted protein during submerged growth. By harnessing gene coexpression network data, we bioinformatically predicted six morphology and productivity associated 'morphogenes', and placed them under control of a conditional Tet-on gene switch using CRISPR-Cas genome editing. Strains were phenotypically screened on solid and liquid media following titration of morphogene expression, generating quantitative measurements of growth rate, filamentous morphology, response to various abiotic perturbations, Euclidean parameters of submerged macromorphologies, and total secreted protein. These data were built into a multiple linear regression model, which identified radial growth rate and fitness under heat stress as positively correlated with protein titres. In contrast, diameter of submerged pellets and cell wall integrity were negatively associated with productivity. Remarkably, our model predicts over 60% of variation in A. niger secreted protein titres is dependent on these four variables, suggesting that they play crucial roles in productivity and are high priority processes to be targeted in future engineering programs. Additionally, this study suggests A. niger dlpA and crzA genes are promising new leads for enhancing protein titres during fermentation. CONCLUSIONS: Taken together this study has identified several potential genetic leads for maximizing protein titres, delivered a suite of chassis strains with user controllable macromorphologies during pilot fermentation studies, and has quantified four crucial factors which impact secreted protein titres in A. niger.

Inducible Knockout of 14-3-3ß Attenuates Proliferation and Spheroid Formation in a Human Glioblastoma Cell Line U87MG.

Glioblastomas (GBs) are the most common and malignant brain tumors in adults. A protein encoded by the gene YWHAB, 14-3-3ß, is commonly found to be upregulated throughout the initiation and progression of GB. The 14-3-3ß has oncogenic roles in several different types of cancer cells through interactions with proteins such as Bad, FBI1, Raf-1, Cdc25b, and others. Previous RNA interference studies have shown that 14-3-3ß promotes proliferation, cell cycle progression, and migration and invasion of GB cells. However, despite the many oncogenic functions of 14-3-3ß, a CRISPR/Cas9 knockout model of 14-3-3ß has not been investigated. This study confirmed previous findings and showed that siRNA inhibition of 14-3-3ß results in reduced cellular proliferation in a human glioblastoma cell line, U87MG. We also used a YWHAB Tet-On CRISPR/Cas9 U87MG cell line that, upon doxycycline induction, leads to robust Cas9 expression and subsequent knockout of 14-3-3ß. Using this model, we show that loss of 14-3-3ß significantly reduces cellular proliferation and spheroid formation of U87MG cells.

Promoting the Differentiation of Neural Progenitor Cells into Oligodendrocytes through the Induction of Olig2 Expression: A Transcriptomic Study Using RNA-seq Analysis.

Oligodendrocytes are the myelinating cells of the central nervous system that facilitate efficient signal transduction. The loss of these cells and the associated myelin sheath can lead to profound functional deficits. Moreover, oligodendrocytes also play key roles in mediating glial-neuronal interactions, which further speaks to their importance in health and disease. Neural progenitor cells (NPCs) are a promising source of cells for the treatment of oligodendrocyte-related neurological diseases due to their ability to differentiate into a variety of cell types, including oligodendrocytes. However, the efficiency of oligodendrocyte differentiation is often low. In this study, we induced the expression of the Olig2 transcription factor in tripotent NPCs using a doxycycline-inducible promoter, such that the extent of oligodendrocyte differentiation could be carefully regulated. We characterized the differentiation profile and the transcriptome of these inducible oligodendrogenic NPCs (ioNPCs) using a combination of qRT-PCR, immunocytochemistry and RNA sequencing with gene ontology (GO) and gene set enrichment analysis (GSEA). Our results show that the ioNPCs differentiated into a significantly greater proportion of oligodendrocytes than the NPCs. The induction of Olig2 expression was also associated with the upregulation of genes involved in oligodendrocyte development and function, as well as the downregulation of genes involved in other cell lineages. The GO and GSEA analyses further corroborated the oligodendrocyte specification of the ioNPCs.

Immortalization Reversibility in the Context of Cell Therapy Biosafety.

Immortalization (genetically induced prevention of replicative senescence) is a promising approach to obtain cellular material for cell therapy or for bio-artificial organs aimed at overcoming the problem of donor material shortage. Immortalization is reversed before cells are used in vivo to allow cell differentiation into the mature phenotype and avoid tumorigenic effects of unlimited cell proliferation. However, there is no certainty that the process of de-immortalization is 100% effective and that it does not cause unwanted changes in the cell. In this review, we discuss various approaches to reversible immortalization, emphasizing their advantages and disadvantages in terms of biosafety. We describe the most promising approaches in improving the biosafety of reversibly immortalized cells: CRISPR/Cas9-mediated immortogene insertion, tamoxifen-mediated self-recombination, tools for selection of successfully immortalized cells, using a decellularized extracellular matrix, and ensuring post-transplant safety with the use of suicide genes. The last process may be used as an add-on for previously existing reversible immortalized cell lines.

A spatiotemporally controlled recombinant cccDNA mouse model for studying HBV and developing drugs against the virus.

Covalently closed circular (ccc) DNA is the template for hepatitis B virus (HBV) replication. The lack of small animal models for characterizing chronic HBV infection has hampered research progress in HBV pathogenesis and drug development. Here, we generated a spatiotemporally controlled recombinant cccDNA (rcccDNA) mouse model by combining Cre/loxP-mediated DNA recombination with the liver-specific "Tet-on/Cre" system. The mouse model harbors three transgenes: a single copy of the HBV genome (integrated at the Rosa26 locus, RHBV), H11-albumin-rtTA (spatiotemporal conditional module), and (tetO)7-Cre (tetracycline response element), and is named as RHTC mouse. By supplying the RHTC mice with doxycycline (DOX)-containing drinking water for two days, the animals generate rcccDNA in hepatocytes, and the rcccDNA supports active HBV gene expression and can maintain HBV viremia persistence for over 60 weeks. Persistent HBV gene expression induces intrahepatic inflammation, fibrosis, and dysplastic pathology, which closely mirrors the disease progression in clinical patients. Bepirovirsen, an antisense oligonucleotide (ASO) targeting all HBV RNA species, showed dose-dependent antiviral effects in the RHTC mouse model. The spatiotemporally controlled rcccDNA mouse is convenient and reliable, providing versatile small animal model for studying cccDNA-centric HBV biology as well as evaluating antiviral therapeutics.

A Ubiquitous Chromatin Opening Element and DNA Demethylation Facilitate Doxycycline-Controlled Expression during Differentiation and in Transgenic Mice.

Synthetic expression cassettes provide the ability to control transgene expression in experimental animal models through external triggers, enabling the study of gene function and the modulation of endogenous regulatory networks in vivo. The performance of synthetic expression cassettes in transgenic animals critically depends on the regulatory properties of the respective chromosomal integration sites, which are affected by the remodeling of the chromatin structure during development. The epigenetic status may affect the transcriptional activity of the synthetic cassettes and even lead to transcriptional silencing, depending on the chromosomal sites and the tissue. In this study, we investigated the influence of the ubiquitous chromosome opening element (UCOE) HNRPA2B1-CBX3 and its subfragments A2UCOE and CBX3 on doxycycline-controlled expression modules within the chromosomal Rosa26 locus. While HNRPA2B1-CBX3 and A2UCOE reduced the expression of the synthetic cassettes in mouse embryonic stem cells, CBX3 stabilized the expression and facilitated doxycycline-controlled expression after in vitro differentiation. In transgenic mice, the CBX3 element protected the cassettes from overt silencing although the expression was moderate and only partially controlled by doxycycline. We demonstrate that CBX3-flanked synthetic cassettes can be activated by decitabine-mediated blockade of DNA methylation or by specific recruitment of the catalytic demethylation domain of the ten-eleven translocation protein TET1 to the synthetic promoter. This suggests that CBX3 renders the synthetic cassettes permissive for subsequent epigenetic activation, thereby supporting doxycycline-controlled expression. Together, this study reveals a strategy for overcoming epigenetic constraints of synthetic expression cassettes, facilitating externally controlled transgene expression in mice.

Tet-Regulated Expression and Optical Clearing for In Vivo Visualization of Genetically Encoded Chimeric dCas9/Fluorescent Protein Probes.

The catalytically inactive mutant of Cas9 (dCas9) endonuclease has multiple biomedical applications, with the most useful being the activation/repression of transcription. dCas9 family members are also emerging as potential experimental tools for gene mapping at the level of individual live cells and intact tissue. We performed initial testing on a set of tools for Cas9-mediated visualization of nuclear compartments. We investigated doxycycline (Dox)-inducible (Tet-On) intracellular distribution of constructs encoding dCas9 orthologs from St. thermophilus (St) and N. meningitides (Nm) fused with EGFP and mCherry fluorescent proteins (FP) in human A549 cells. We also studied time-dependent expression of these chimeric fluorescent constructs (dCas9-FP) after Tet-On induction in live cells and compared it with the time course of dCas9-FP expression in experimental dCas9-FP-expressing tumor xenografts using a combination of fluorescence imaging and in vivo contrast-assisted magnetic resonance imaging for assessing the extent of tumor perfusion. In vivo Dox-induction of mCherry-chimera expression occurred in tumor xenografts as early as 24 h post-induction and was visualized by using optical clearing (OC) of the skin. OC via topical application of gadobutrol enabled high-contrast imaging of FP expression in tumor xenografts due to a 1.1-1.2-fold increase in FI in both the red and green channels.

An optimized Tet-On system for conditional control of gene expression in sea urchins.

Sea urchins and other echinoderms are important experimental models for studying developmental processes. The lack of approaches for conditional gene perturbation, however, has made it challenging to investigate the late developmental functions of genes that have essential roles during early embryogenesis and genes that have diverse functions in multiple tissues. The doxycycline-controlled Tet-On system is a widely used molecular tool for temporally and spatially regulated transgene expression. Here, we optimized the Tet-On system to conditionally induce gene expression in sea urchin embryos. Using this approach, we explored the roles the MAPK signaling plays in skeletogenesis by expressing genes that perturb the pathway specifically in primary mesenchyme cells during later stages of development. We demonstrated the wide utility of the Tet-On system by applying it to a second sea urchin species and in cell types other than the primary mesenchyme cells. Our work provides a robust and flexible platform for the spatiotemporal regulation of gene expression in sea urchins, which will considerably enhance the utility of this prominent model system.

Establishment of leukemia cell model with inducible AML1-ETO expression and its effect on fatty acid metabolism in leukemia cells / 中华血液学杂志

Objective: To investigate the effect of the AML1-ETO (AE) fusion gene on the biological function of U937 leukemia cells by establishing a leukemia cell model that induces AE fusion gene expression. Methods: The doxycycline (Dox) -dependent expression of the AE fusion gene in the U937 cell line (U937-AE) were established using a lentivirus vector system. The Cell Counting Kit 8 methods, including the PI and sidanilide induction, were used to detect cell proliferation, cell cycle-induced differentiation assays, respectively. The effect of the AE fusion gene on the biological function of U937-AE cells was preliminarily explored using transcriptome sequencing and metabonomic sequencing. Results: ①The Dox-dependent Tet-on regulatory system was successfully constructed to regulate the stable AE fusion gene expression in U937-AE cells. ②Cell proliferation slowed down and the cell proliferation rate with AE expression (3.47±0.07) was lower than AE non-expression (3.86 ± 0.05) after inducing the AE fusion gene expression for 24 h (P<0.05). The proportion of cells in the G(0)/G(1) phase in the cell cycle increased, with AE expression [ (63.45±3.10) %) ] was higher than AE non-expression [ (41.36± 9.56) %] (P<0.05). The proportion of cells expressing CD13 and CD14 decreased with the expression of AE. The AE negative group is significantly higher than the AE positive group (P<0.05). ③The enrichment analysis of the transcriptome sequencing gene set revealed significantly enriched quiescence, nuclear factor kappa-light-chain-enhancer of activated B cells, interferon-α/γ, and other inflammatory response and immune regulation signals after AE expression. ④Disorder of fatty acid metabolism of U937-AE cells occurred under the influence of AE. The concentration of the medium and short-chain fatty acid acylcarnitine metabolites decreased in cells with AE expressing, propionyl L-carnitine, wherein those with AE expression (0.46±0.13) were lower than those with AE non-expression (1.00±0.27) (P<0.05). The metabolite concentration of some long-chain fatty acid acylcarnitine increased in cells with AE expressing tetradecanoyl carnitine, wherein those with AE expression (1.26±0.01) were higher than those with AE non-expression (1.00±0.05) (P<0.05) . Conclusion: This study successfully established a leukemia cell model that can induce AE expression. The AE expression blocked the cell cycle and inhibited cell differentiation. The gene sets related to the inflammatory reactions was significantly enriched in U937-AE cells that express AE, and fatty acid metabolism was disordered.

Enhancement of fatty acid degradation pathway promoted glucoamylase synthesis in Aspergillus niger.

BACKGROUND: Our recent multi-omics analyses of glucoamylase biosynthesis in Aspergillus niger (A. niger) suggested that lipid catabolism was significantly up-regulated during high-yield period under oxygen limitation. Since the catabolism of fatty acids can provide energy compounds such as ATP and important precursors such as acetyl-CoA, we speculated that enhancement of this pathway might be beneficial to glucoamylase overproduction. RESULTS: Based on previous transcriptome data, we selected and individually overexpressed five candidate genes involved in fatty acid degradation under the control of the Tet-on gene switch in A. niger. Overexpression of the fadE, fadA and cyp genes increased the final specific enzyme activity and total secreted protein on shake flask by 21.3 ~ 31.3% and 16.0 ~ 24.2%, respectively. And a better inducible effect by doxycycline was obtained from early logarithmic growth phase (18 h) than stationary phase (42 h). Similar with flask-level results, the glucoamylase content and total extracellular protein in engineered strains OE-fadE (overexpressing fadE) and OE-fadA (overexpressing fadA) on maltose-limited chemostat cultivation were improved by 31.2 ~ 34.1% and 35.1 ~ 38.8% compared to parental strain B36. Meanwhile, intracellular free fatty acids were correspondingly decreased by 41.6 ~ 44.6%. The metabolomic analysis demonstrated intracellular amino acids pools increased 24.86% and 18.49% in two engineered strains OE-fadE and OE-fadA compared to B36. Flux simulation revealed that increased ATP, acetyl-CoA and NADH was supplied into TCA cycle to improve amino acids synthesis for glucoamylase overproduction. CONCLUSION: This study suggested for the first time that glucoamylase production was significantly improved in A. niger by overexpression of genes fadE and fadA involved in fatty acids degradation pathway. Harnessing the intracellular fatty acids could be a strategy to improve enzyme production in Aspergillus niger cell factory.

ERCC1 Overexpression Increases Radioresistance in Colorectal Cancer Cells.

Preoperative concurrent chemoradiotherapy (CCRT) is a standard treatment for locally advanced rectal cancer patients, but 20-30% do not benefit from the desired therapeutic effects. Previous reports indicate that high levels of ERCC1 reduce the effectiveness of cisplatin-based CCRT; however, it remains unclear as to whether ERCC1 overexpression increases radiation resistance. To clarify the correlation between ERCC1 levels and radiation (RT) resistance, we established two cell lines (HCT116-Tet-on and COLO205-Tet-on), induced them to overexpress ERCC1, detected cell survival following exposure to radiation, established HCT116-Tet-on and COLO205-Tet-on heterotopic cancer animal models, and detected tumor volume following exposure to radiation. We found that ERCC1 overexpression increased radiation resistance. After regulating ERCC1 levels and radiation exposure to verify the correlation, we noted that increased radiation resistance was dependent on ERCC1 upregulation in both cell lines. For further verification, we exposed HCT116-Tet-on and COLO205-Tet-on heterotopic cancer animal models to radiation and observed that ERCC1 overexpression increased colorectal cancer tumor radioresistance in both. Combined, our results suggest that ERCC1 overexpression may serve as a suitable CCRT prognostic marker for colorectal cancer patients.

Efficient and reversible Cas13d-mediated knockdown with an all-in-one lentivirus-vector.

Type VI CRISPR effector Cas13d from Ruminococcus flavefaciens XPD3002 (RfxCas13d) is an RNA-guided RNA endonuclease. RfxCas13d has been harnessed to knockdown gene expression with high specificity in various systems including mammalian cells. While inducible knockdown is advantageous over constitutive knockdown in many scenarios, current inducible systems of RfxCas13d express CRISPR RNA and Cas13d separately. Such systems could be cumbersome to handle and may hamper the application of RfxCas13d in some scenarios. Here, we design an all-in-one Cas13d lentivirus vector which renders efficient and inducible knockdown in a doxycycline dosage-dependent manner. Furthermore, we find that Cas13d has a short half-life in mammalian cells. As a result, knockdown can be promptly reversed after doxycycline withdrawal. This vector is particularly useful for applications involving indispensable genes and/or in cells hard to transduce.

Parabolic relationship between SMAD3 expression level and the reprogramming efficiency of goat induced mammary epithelial cells.

Mammary epithelial cells are the only cells of mammary glands with lactation capacity. They are closely related to mammary development and milk yield. Our earlier studies showed that the transformation of goat fibroblasts into induced mammary epithelial cells (iMECs) was closely correlated with SMAD3 overexpression. Therefore, we further explored the role of SMAD3 on iMECs reprogramming in this study. The SMAD3 gene was overexpressed in goat ear fibroblasts using the tetracycline-induced expression method. The outcomes demonstrated that goat ear fibroblasts can be converted into iMECs by overexpressing the SMAD3 gene. In contrast, it was discovered that SMAD3 downregulation by RNA interference significantly decrease the reprogramming efficiency of iMECs. These results show that SMAD3 plays a key regulatory role in the reprogramming of iMECs. Surprisingly, we also found a parabolic relationship between SMAD3 expression level and iMECs reprogramming efficiency, and that the reprogramming efficiency was maximum when the addition of doxycycline concentration was 5 µg/ml. In light of this, our findings may offer new perspectives on the regulatory mechanism governing mammary epithelial cell fate in goats as well as a fresh approach to studying mammary development and differentiation in vitro.