Generation and utilization of endostatin-sensitive cell lines for assessing the biological activity of endostatin.

Recombinant proteins are gaining increasing popularity for treating human diseases. The clinical effectiveness of recombinant proteins is directly related to their biological activity, which is an important indicator in drug development and quality control. However, certain recombinant proteins have unclear or complex signal pathways, making detecting their activity in vitro difficult. For instance, recombinant human endostatin (endostatin), a new antitumor drug developed in China, lacks a sensitive and stable assay for its biological activity since being market approval. To address this issue, we performed a genome-wide screening of immortalized human umbilical vein endothelial cells (HUVECs) using a CRISPR/Cas9 knockout library containing 20,000 targeted genes. We identified two potential endostatin-resistant genes, NEPSPP and UTS2, and successfully constructed a highly sensitive cell line, HUVEC-UTS2-3#, by knocking down the UTS2 gene. Based on the optimized parameters of HUVEC-UTS2-3# cells, we established a new method for detecting the biological activity of endostatin. The method was validated, and it produced results consistent with primary HUVEC cells but with higher sensitivity and more stable data. The use of gene-editing technology provides a novel solution for detecting the biological activity of recombinant proteins that other methods cannot detect.

A genome-wide CRISPR screen identified host genes essential for intracellular Brucella survival.

Brucella is a zoonotic intracellular bacterium that poses threats to human health and economic security. Intracellular infection is a hallmark of the agent Brucella and a primary cause of distress, through which the bacterium regulates the host intracellular environment to promote its own colonization and replication, evading host immunity and pharmaceutical killing. Current studies of Brucella intracellular processes are typically premised on bacterial phenotype such as intracellular bacterial survival, followed by biochemical or molecular biological approaches to reveal detailed mechanisms. While such processes can deepen the understanding of Brucella-host interaction, the insights into host alterations in infection would be easily restricted to known pathways. In the current study, we applied CRISPR Cas9 screen to identify host genes that are most affected by Brucella infection on cell viability at the genomic level. As a result of CRISPR screening, we firstly identified that knockout of the negatively selected genes GOLGA6L6, DEFB103B, OR4F29, and ERCC6 attenuate the viability of both the host cells and intracellular Brucella, suggesting these genes to be potential therapeutic targets for Brucella control. In particular, knockout of DEFB103B diminished Brucella intracellular survival by altering host cell autophagy. Conversely, knockout of positive screening genes promoted intracellular proliferation of Brucella. In summary, we screened host genes at the genomic level throughout Brucella infection, identified host genes that are previously not recognized to be involved in Brucella infection, and provided targets for intracellular infection control.IMPORTANCEBrucella is a Gram-negative bacterium that infects common mammals causing arthritis, myalgia, neuritis, orchitis, or miscarriage and is difficult to cure with antibiotics due to its intracellular parasitism. Therefore, unraveling the mechanism of Brucella-host interactions will help controlling Brucella infections. CRISPR-Cas9 is a gene editing technology that directs knockout of individual target genes by guided RNA, from which genome-wide gene-knockout cell libraries can be constructed. Upon infection with Brucella, the cell library would show differences in viability as a result of the knockout and specific genes could be revealed by genomic DNA sequencing. As a result, genes affecting cell viability during Brucella infection were identified. Further testing of gene function may reveal the mechanisms of Brucella-host interactions, thereby contributing to clinical therapy.

Development and Immunological Evaluation of a Multiantigen Thermostable Nanovaccine Adjuvanted with T-Cell-Activating Scaffold for African Swine Fever.

African swine fever is an acute and highly contagious infectious disease with a mortality rate of up to 100%. The lack of commercial vaccines and drugs is a serious economic threat to the global pig industry. Cell-mediated immunity plays an essential role in protection against viral infection. We previously reported the rational design of a T-cell-activating thermostable scaffold (RPT) for antigen delivery and improved cellular immunity. We conjugated antigens P30, P54, P72, CD2 V, and CP312R to RPT, using a SpyCatcher/SpyTag covalent attachment strategy to construct nanovaccines (multiantigens-RPT). Multiantigens-RPT exhibited significantly higher thermal, storage, and freeze-thaw stability. The specific antibodies IgG and IgG2a of the multiantigen-RPT-immunized were higher than the antigens cocktail-immunized by approximately 10-100 times. ELISpot demonstrated that more IFN-γ-secreting cells were produced by the multiantigen-RPT-immunized than by the antigens cocktail-immunized. Delivery of the multiantigen nanovaccine by a T-cell-activating scaffold induced strong humoral and cellular immune responses in mice and pigs and is a potentially useful candidate vaccine for the African swine fever virus.

SMURF1 controls the PPP3/calcineurin complex and TFEB at a regulatory node for lysosomal biogenesis.

Macroautophagy/autophagy is a homeostatic process in response to multiple signaling, such as the lysosome-dependent recycling process of cellular components. Starvation-induced MTOR inactivation and PPP3/calcineurin activation were shown to promote the nuclear translocation of TFEB. However, the mechanisms via which signals from endomembrane damage are transmitted to activate PPP3/calcineurin and orchestrate autophagic responses remain unknown. This study aimed to show that autophagy regulator SMURF1 controlled TFEB nuclear import for transcriptional activation of the lysosomal biogenesis. We showed that blocking SMURF1 affected lysosomal biogenesis in response to lysosomal damage by preventing TFEB nuclear translocation. It revealed galectins recognized endolysosomal damage, and led to recruitment of SMURF1 and the PPP3/calcineurin apparatus on lysosomes. SMURF1 interacts with both LGALS3 and PPP3CB to form the LGALS3-SMURF1-PPP3/calcineurin complex. Importantly, this complex further stabilizes TFEB, thereby activating TFEB for lysosomal biogenesis. We determined that LLOMe-mediated TFEB nuclear import is dependent on SMURF1 under the condition of MTORC1 inhibition. In addition, SMURF1 is required for PPP3/calcineurin activity as a positive regulator of TFEB. SMURF1 controlled the phosphatase activity of the PPP3CB by promoting the dissociation of its autoinhibitory domain (AID) from its catalytic domain (CD). Overexpression of SMURF1 showed similar effects as the constitutive activation of PPP3CB. Thus, SMURF1, which bridges environmental stress with the core autophagosomal and autolysosomal machinery, interacted with endomembrane sensor LGALS3 and phosphatase PPP3CB to control TFEB activation.Abbreviations: ATG: autophagy-related; LLOMe: L-Leucyl-L-Leucine methyl ester; ML-SA1: mucolipin synthetic agonist 1; MTOR: mechanistic target of rapamycin kinase; PPP3CB: protein phosphatase 3 catalytic subunit beta; RPS6KB1/p70S6K: ribosomal protein S6 kinase B1; SMURF1: SMAD specific E3 ubiquitin protein ligase 1; TFEB: transcription factor EB.

Thermostable T Cell Multiepitope Nanoparticle Antigens Inducing Potent Immune Responses against the Swine Fever Virus.

African swine fever (ASF) is caused by the African swine fever virus (ASFV) and is a highly contagious, acute, febrile disease that has high morbidity and mortality rates in domestic and wild swine. However, a safe and effective vaccine against ASF remains unavailable as single antigens fail to provide sufficient protection. Therefore, a combination of multiple antigens with an efficient delivery system might be an alternative strategy. Herein, a de novo-designed antigen with multiple T-cell epitopes (TEPs) of ASFV was conjugated for surface display on self-assembled nanoparticles (NPs) of Aquifex aeolicus lumazine synthase (AaLS) and Quasibacillus thermotolerans encapsulin (QT) through the SpyCatcher/SpyTag system to construct nanovaccines (TEP-Spy-NPs). TEP-Spy-NPs exhibited significantly more thermal, storage, and freeze-thaw stability in comparison to TEP monomers. TEP-Spy-NPs were highly immunogenic and induced strong polyclonal antibody responses in mice and pigs. The specific antibody titers against the TEP of the TEP-Spy-AaLS and TEP-Spy-QT groups were significantly higher than those of the TEP monomer immune group after the second booster immunization. The antibody titer against TEP of the TEP-Spy-QT group was approximately twice that of the TEP-Spy-AaLS group in mice. ELISpot analysis demonstrated that more IFN-γ- and IL-2-secreting splenic lymphocytes were produced by TEP-Spy-AaLS- and TEP-Spy-QT-immunized mice than by TEP monomer-immunized mice. TEP-Spy-NPs elicited stronger cellular immunity and in vivo immunity in immunized pigs than did TEP monomers. Thus, the TEP nanovaccine successfully induced strong humoral and cellular immune responses in mice and pigs, and TEP-Spy-NPs have the potential as candidate vaccines for ASFV.

A replicative recombinant HPV16 E7 expression virus upregulates CD36 in C33A cells.

Objective: In past decades, the role of high-risk HPV (HR-HPV) infection in cancer pathogenesis has been extensively studied. The viral E7 protein expressed in pre-malignant cells has been identified as an ideal target for immunological intervention. However, the cultivation of HPV in vitro remains a significant challenge, as well as the lack of methods for expressing the HPV E7 protein and generating replication-competent recombinant viral particles, which posed a major obstacle to further exploration of the function and carcinogenic mechanisms of the E7 oncoprotein. Therefore, it is imperative to investigate novel methodologies to construct replication-competent recombinant viral particles that express the HPV E7 protein to facilitate the study of its function. Methods: We initiated the construction of recombinant viral particles by utilizing the ccdB-Kan forward/reverse screening system in conjunction with the Red/ExoCET recombinant system. We followed the infection of C33A cells with the obtained recombinant virus to enable the continuous expression of HPV16 E7. Afterwards, the total RNA was extracted and performed transcriptome sequencing using RNA-Seq technology to identify differentially expressed genes associated with HPV-induced oncogenicity. Results: We successfully established replicative recombinant viral particles expressing HPV16 E7 stably and continuously. The C33A cells were infected with recombinant viral particles to achieve overexpression of the E7 protein. Subsequently, RNA-Seq analysis was conducted to assess the changes in host cell gene expression. The results revealed an upregulation of the CD36 gene, which is associated with the HPV-induced oncogenic pathways, including PI3K-Akt and p53 signaling pathway. qRT-PCR analysis further identified that the upregulation of the CD36 gene due to the expression of HPV16 E7. Conclusion: The successful expression of HPV16 E7 in cells demonstrates that the replicated recombinant virus retains the replication and infection abilities of Ad4, while also upregulating the CD36 gene involved in the PI3K-Akt signaling and p53 pathways, thereby promoting cell proliferation. The outcome of this study provides a novel perspective and serves as a solid foundation for further exploration of HPV-related carcinogenesis and the development of replicative HPV recombinant vaccines capable of inducing protective immunity against HPV.

Loss of Ptpmt1 limits mitochondrial utilization of carbohydrates and leads to muscle atrophy and heart failure in tissue-specific knockout mice.

While mitochondria in different tissues have distinct preferences for energy sources, they are flexible in utilizing competing substrates for metabolism according to physiological and nutritional circumstances. However, the regulatory mechanisms and significance of metabolic flexibility are not completely understood. Here, we report that the deletion of Ptpmt1, a mitochondria-based phosphatase, critically alters mitochondrial fuel selection - the utilization of pyruvate, a key mitochondrial substrate derived from glucose (the major simple carbohydrate), is inhibited, whereas the fatty acid utilization is enhanced. Ptpmt1 knockout does not impact the development of the skeletal muscle or heart. However, the metabolic inflexibility ultimately leads to muscular atrophy, heart failure, and sudden death. Mechanistic analyses reveal that the prolonged substrate shift from carbohydrates to lipids causes oxidative stress and mitochondrial destruction, which in turn results in marked accumulation of lipids and profound damage in the knockout muscle cells and cardiomyocytes. Interestingly, Ptpmt1 deletion from the liver or adipose tissue does not generate any local or systemic defects. These findings suggest that Ptpmt1 plays an important role in maintaining mitochondrial flexibility and that their balanced utilization of carbohydrates and lipids is essential for both the skeletal muscle and the heart despite the two tissues having different preferred energy sources.

Cells are powered by mitochondria, a group of organelles that produce chemical energy in the form of molecules called ATP. This energy is derived from the breakdown of carbohydrates, fats, and proteins. The number of mitochondria in a cell and the energy source they use to produce ATP varies depending on the type of cell. Mitochondria can also switch the molecules they use to produce energy when the cell is responding to stress or disease. The heart and the skeletal muscles ­ which allow movement ­ are two tissues that require large amounts of energy, but it remained unknown whether disrupting mitochondrial fuel selection affects how these tissues work. To answer these questions, Zheng, Li, Li et al. investigated the role of an enzyme found in mitochondria called Ptpmt1. Genetically deleting Ptpmt1 in the heart and skeletal muscle of mice showed that while the development of these organs was not affected, mitochondria in these cells switched from using carbohydrates to using fats as an energy source. Over time, this shift damaged both the mitochondria and the tissues, leading to muscle wasting, heart failure, and sudden death in the mice. This suggests that balanced use of carbohydrates and fats is essential for the muscles and heart. These findings imply that long-term use of medications that alter the fuel that mitochondria use may be detrimental to patients' health and could cause heart dysfunction. This may be important for future drug development, as well as informing decisions about medication taken in the clinic.

Chrysanthemum sporopollenin: A novel vaccine delivery system for nasal mucosal immunity.

Objective: Mucosal immunization was an effective defender against pathogens. Nasal vaccines could activate both systemic and mucosal immunity to trigger protective immune responses. However, due to the weak immunogenicity of nasal vaccines and the lack of appropriate antigen carriers, very few nasal vaccines have been clinically approved for human use, which was a major barrier to the development of nasal vaccines. Plant-derived adjuvants are promising candidates for vaccine delivery systems due to their relatively safe immunogenic properties. In particular, the distinctive structure of pollen was beneficial to the stability and retention of antigen in the nasal mucosa. Methods: Herein, a novel wild-type chrysanthemum sporopollenin vaccine delivery system loaded with a w/o/w emulsion containing squalane and protein antigen was fabricated. The unique internal cavities and the rigid external walls within the sporopollenin skeleton construction could preserve and stabilize the inner proteins. The external morphological characteristics were suitable for nasal mucosal administration with high adhesion and retention. Results: Secretory IgA antibodies in the nasal mucosa can be induced by the w/o/w emulsion with the chrysanthemum sporopollenin vaccine delivery system. Moreover, the nasal adjuvants produce a stronger humoral response (IgA and IgG) compared to squalene emulsion adjuvant. Mucosal adjuvant benefited primarily from prolongation of antigens in the nasal cavity, improvement of antigen penetration in the submucosa and promotion of CD8+ T cells in spleen. Disccusion: Based on effective delivering both the adjuvant and the antigen, the increase of protein antigen stability and the realization of mucosal retention, the chrysanthemum sporopollenin vaccine delivery system has the potential to be a promising adjuvant platform. This work provide a novel idea for the fabrication of protein-mucosal delivery vaccine.

Bacterial cellulose flakes loaded with Bi2MoO6 nanoparticles and quantum dots for the photodegradation of antibiotic and dye pollutants.

Effective strategies to improve charge separation in semiconductor particles are critical for improving the photodegradation of organic pollutants at levels sufficient for environmental applications. Herein, Bi2MoO6 (BMOMOF), comprising both nanoparticles (NPs) and quantum dots (QDs), was synthesized from a bismuth-based metal-organic framework (Bi-MOF) precursor. Surface defects on BMOMOF, the combination of NPs and QDs, and modified energy band edges improved photogenerated charge separation and facilitated redox reactions. When compared to BMO derived from uncoordinated Bi, the BMOMOF photocatalyst (PC) was more efficient at photodegrading tetracycline hydrochloride (TCH) and ciprofloxacin (CIP), two widely-used antibiotics ubiquitous in wastewater, as well as the carcinogenic pollutant rhodamine B (RhB). BMOMOF was then loaded on the biopolymer bacterial cellulose (BC) to further enhance photocatalytic performance and facilitate the recovery of the PC after water treatment processes. The novel BMOMOF/BC photocatalytic flakes were significantly larger than pure BMOMOF, and thus easier to recuperate. Furthermore, anchoring BMOMOF on BC flakes augmented significantly the photodegradation of TCH, CIP, and RhB, mainly because hydroxyl groups in BC act as hole traps facilitating photogenerated electron-hole separation. Results obtained with BMOMOF/BC highlight promising approaches to develop optimal PCs for aqueous pollutants degradation.

Development of an Adeno-Associated Virus-Vectored SARS-CoV-2 Vaccine and Its Immunogenicity in Mice.

Owing to the outbreak of the novel coronavirus (SARS-CoV-2) worldwide at the end of 2019, the development of a SARS-CoV-2 vaccine became an urgent need. In this study, we developed a type 9 adeno-associated virus vectored vaccine candidate expressing a dimeric receptor binding domain (RBD) of the SARS-CoV-2 spike protein (S protein) and evaluated its immunogenicity in a murine model. The vaccine candidate, named AAV9-RBD virus, was constructed by inserting a signal peptide to the N-terminus of two copies of RBD, spaced by a linker, into the genome of a type 9 adeno-associated virus. In vitro assays showed that HeLa cells infected by the recombinant AAV virus expressed high levels of the recombinant RBD protein, mostly found in the cell culture supernatant. The recombinant AAV9-RBD virus was cultured and purified. The genome titer of the purified recombinant AAV9-RBD virus was determined to be 2.4 × 1013 genome copies/mL (GC/mL) by Q-PCR. Balb/c mice were immunized with the virus by intramuscular injection or nasal drip administration. Eight weeks after immunization, neutralizing antibodies against the new coronavirus pseudovirus were detected in the sera of all mice; the mean neutralizing antibody EC50 values were 517.7 ± 292.1 (n=10) and 682.8 ± 454.0 (n=10) in the intramuscular injection group and nasal drip group, respectively. The results of this study showed that the recombinant AAV9-RBD virus may be used for the development of a SARS-CoV-2 vaccine.

Gold-viral particle identification by deep learning in wide-field photon scattering parametric images.

The ability to identify virus particles is important for research and clinical applications. Because of the optical diffraction limit, conventional optical microscopes are generally not suitable for virus particle detection, and higher resolution instruments such as transmission electron microscopy (TEM) and scanning electron microscopy (SEM) are required. In this paper, we propose a new method for identifying virus particles based on polarization parametric indirect microscopic imaging (PIMI) and deep learning techniques. By introducing an abrupt change of refractivity at the virus particle using antibody-conjugated gold nanoparticles (AuNPs), the strength of the photon scattering signal can be magnified. After acquiring the PIMI images, a deep learning method was applied to identify discriminating features and classify the virus particles, using electron microscopy (EM) images as the ground truth. Experimental results confirm that gold-virus particles can be identified in PIMI images with a high level of confidence.

Preclinical Application of Conditional Reprogramming Culture System for Laryngeal and Hypopharyngeal Carcinoma.

Management of laryngeal and hypopharyngeal squamous cell carcinoma (LHSCC) remains highly challenging due to highly variable therapeutic responses. By establishing an in vitro model for LHSCC based on conditional reprogramming (CR), a cell-culture technique, we aim to investigate its potential value on personalized cancer therapies. Herein, a panel of 28 human LHSCC CR cells were established from 50 tumor tissues using the CR method. They retained tumorigenic potential upon xenotransplantation and recapitulated molecular characteristics of LHSCC. Differential responses to anticancer drugs and radiotherapy were detected in vitro. CR cells could be transformed to xenograft and organoid, and they shared comparable drug responses. The clinical drug responses were consistent with in vitro drug responses. Collectively, the patient-derived CR cell model could promisingly be utilized in clinical decision-making and assisted in the selection of personalized therapies for LHSCC.

Identification of the Prognostic Signatures of Glioma With Different PTEN Status.

The high-grade glioma is characterized by cell heterogeneity, gene mutations, and poor prognosis. The deletions and mutations of the tumor suppressor gene PTEN (5%-40%) in glioma patients are associated with worse survival and therapeutic resistance. Characterization of unique prognosis molecular signatures by PTEN status in glioma is still unclear. This study established a novel risk model, screened optimal prognostic signatures, and calculated the risk score for the individual glioma patients with different PTEN status. Screening results revealed fourteen independent prognostic gene signatures in PTEN-wt and three in the -50PTEN-mut subgroup. Moreover, we verified risk score as an independent prognostic factor significantly correlated with tumor malignancy. Due to the higher malignancy of the PTEN-mut gliomas, we explored the independent prognostic signatures (CLCF1, AEBP1, and OS9) for a potential therapeutic target in PTEN-mut glioma. We further separated IDH wild-type glioma patients into GBM and LGG to verify the therapeutic target along with PTEN status, notably, the above screened therapeutic targets are also significant prognostic genes in both IDH-wt/PTEN-mut GBM and LGG patients. We further identified the small molecule compound (+)-JQ1 binds to all three targets, indicating a potential therapy for PTEN-mut glioma. In sum, gene signatures and risk scores in the novel risk model facilitate glioma diagnosis, prognosis prediction, and treatment.

Feedback activation of STAT3 limits the response to PI3K/AKT/mTOR inhibitors in PTEN-deficient cancer cells.

The PI3K/AKT/mTOR signaling pathway is constitutively active in PTEN-deficient cancer cells, and its targeted inhibition has significant anti-tumor effects. However, the efficacy of targeted therapies is often limited due to drug resistance. The relevant signaling pathways in PTEN-deficient cancer cells treated with the PI3K/mTOR inhibitor BEZ235 were screened using a phosphokinase array, and further validated following treatment with multiple PI3K/AKT/mTOR inhibitors or AKT knockdown. The correlation between PTEN expression levels and STAT3 kinase phosphorylation in the tissue microarrays of gastric cancer patients was analyzed by immunohistochemistry. Cell proliferation and clonogenic assays were performed on the suitably treated PTEN-deficient cancer cells. Cytokine arrays, small molecule inhibition and knockdown assays were performed to identify related factors. PTEN-deficient tumor xenografts were established in nude mice that were treated with PI3K/AKT/mTOR and/or STAT3 inhibitors. PTEN deficiency was positively correlated with low STAT3 activity. PI3K/mTOR inhibitors increased the expression and secretion of macrophage migration inhibitory factor (MIF) and activated the JAK1/STAT3 signaling pathway. Both cancer cells and in vivo tumor xenografts showed that the combined inhibition of PI3K/AKT/mTOR and STAT3 activity enhanced the inhibitory effect of BEZ235 on the proliferation of PTEN-deficient cancer cells. Our findings provide a scientific basis for a novel treatment strategy in cancer patients with PTEN deficiency.

Label-free sensing of virus-like particles below the sub-diffraction limit by wide-field photon state parametric imaging of a gold nanodot array.

A parallel four-quadrant sensing method utilizing a specially designed gold nanodot array is created for sensing virus-like particles with a sub-diffraction limit size (∼100 nm) in a wide-field image. Direct label-free sensing of viruses using multiple four-quadrant sensing channels in parallel in a wide-field view enables the possibility of high-throughput onsite screening of viruses.

A double-locus scarless genome editing system in Escherichia coli.

OBJECTIVE: To develop a convenient double-locus scarless genome editing system in Escherichia coli, based on the type II Streptococcus pyogenes CRISPR/Cas9 and λ Red recombination cassette. RESULTS: A two-plasmid genome editing system was constructed. The large-sized plasmid harbors the cas9 and λ Red recombination genes (gam, bet, and exo), while the small-molecular plasmid can simultaneously express two different gRNAs (targeting genome RNAs). The recombination efficiency was tested by targeting the galK, lacZ, and dbpA genes in E. coli with ssDNA or dsDNA. Resulting concurrent double-locus recombination efficiencies were 88 ± 5.5% (point mutation), 39.7 ± 4.3% (deletion/insertion), and 57.8 ± 3.4%-58.5 ± 4.1% (mixed point and deletion/insertion mutation), depending on 30 (ssDNA) or 40 bp (dsDNA) homologous side arms employed. In addition, the curing efficiency of the guide plasmid expressing gRNAs for negative selection was higher (96 ± 3% in 4 h) than the help plasmid carrying cas9 and λ Red (92 ± 2% in 9 h). CONCLUSIONS: The new editing system is convenient and efficient for simultaneous double-locus recombination in the genome and should be favorable for high-throughput multiplex genome editing in synthetic biology and metabolic engineering.

Tanshinone I regulates autophagic signaling via the activation of AMP-activated protein kinase in cancer cells.

Tanshinone I, one of the components of Salvia miltiorrhiza Bunge, exhibits anti-tumor ability and induces autophagy. But the mechanisms are not fully understood. This study aims to investigate whether AMP-activated protein kinase dependent pathway is involved in the autophagic signaling regulation and its relationship with tumor suppression. Breast cancer cells (MDA-MB-231, MCF-7) and hepatocellular carcinoma cells (HepG2) were treated with Tanshinone I or vehicle. Acridine orange dyeing and transmission electron microscopy were employed to evaluate autophagic cells. MTT and Cell Counting Kit-8 assays were used to detect the effect of Tanshinone I combined with autophagy inhibitors on cell proliferation. Western blot was used to detect the expression levels of Beclin1 and LC3-I/II, as well as the phosphorylation of AMPKα and ULK1. Our results showed that Tanshinone I suppressed proliferation of HepG2, MDA-MB-231 and MCF-7 cancer cell lines. LC3-II and P62 were induced by Tanshinone I in all three cancer cell lines. But autophagic flux analysis showed that Tanshinone I treatment induced autophagy only in MDA-MB-231, which was also proved by transmission electron microscopy. Tanshinone I upregulated the phosphorylation of AMPKα and its downstream ULK1. AMP-activated protein kinase inhibitor compound C attenuated Beclin 1 and LC3-II expression induced by Tanshinone I in HepG2. In MDA-MB-231, compound C surprisingly induced LC3-II upregulation which is independent of AMPKα activation. Under this circumstance, treatment of Tanshinone I combined with compound C significantly inhibited MDA-MB-231 proliferation, compared with Tanshinone I treatment alone. This study demonstrates that Tanshinone I could induce cancer cell death and regulate autophagy signaling in breast cancer and hepatic carcinoma cells. Activation of AMPKα was found to be involved in autophagic signaling regulation by Tanshinone I.

Tuning chromosomal gene expression in Escherichia coli by combining single-stranded oligonucleotides mediated recombination and kil counter selection system.

Extensively modulating gene expression to achieve optimal flux is a critical step in metabolic engineering. Gene expression is usually modulated at the transcriptional level by controlling the strength of a promoter. However, this type of modulation is often hampered by its inability to fully sample the complete continuum of transcriptional control. In Escherichia coli, this limitation can be solved by constructing promoters with a wide range of strengths. In this study, a highly efficient method was developed to modulate a particular chromosomal gene of E. coli at a wide range of expression levels. This was achieved by combining highly efficient single-stranded oligonucleotide-mediated recombination and a stringent counter selection system kil. Using this strategy, a chromosomal library, with a range from 0.3% to 388% relative to the wild lac promoter, was easily obtained. The strength of our chromosomal promoter library was approximately 5-60 times wider in range than those of libraries reported before.

Feedback Activation of SGK3 and AKT Contributes to Rapamycin Resistance by Reactivating mTORC1/4EBP1 Axis via TSC2 in Breast Cancer.

The mTORC1 inhibitors, such as rapamycin and its analogs, show limited antitumor activity in clinic, reasons for which have not been clearly elucidated. Here, we undertook an effort to uncover the mechanisms underlying the limited efficacy of rapamycin, and found that the transit suppression of 4EBP1 phosphorylation led to cap-dependent translation and cell proliferation in breast cancer cells. AKT only partially contributed to 4EBP1 re-phosphorylation. By taking advantage of mass spectrometry-based phosphoproteomic analysis, we identified SGK3 as a potent kinase involved in 4EBP1 re-phosphorylation. SGK3 deletion inhibited 4EBP1 phosphorylation and cap-dependent translation. Importantly, 4EBP1 phosphorylation was positively correlated with SGK3 activity in 67 clinical breast cancer specimens. Moreover, SGK3 deletion in combination with AKT inhibition almost blocked the 4EBP1 re-phosphorylation that was induced by rapamycin and profoundly enhanced rapamycin-induced growth inhibition in vitro and in an MCF7 breast cancer mouse xenograft model in vivo. Mechanistically, the feedback activation of SGK3 by rapamycin was dependent on hVps34 and mTORC2, and reactivated mTORC1/4EBP1 axis by phosphorylating TSC2. Collectively, our study reveals a critical role of SGK3 in mediating rapamycin resistance, and provides a rationale for targeting SGK3 to improve mTOR-targeted therapies.

Responsive Cells for rhEGF bioassay Obtained through Screening of a CRISPR/Cas9 Library.

Bioassay of recombinant protein products is important tests to ensure protein effectiveness. Some recombinant protein products have no cells used in their bioassay but instead use animal models, while others have no suitable method. Here, we developed a method to obtain responsive cells used in bioassay of proteins. After screening of a CRISPR/Cas9 library, we obtained a responsive cell line that grew faster in the presence of rhEGF (recombinant human epidermal growth factor) than that of control cells. We used this cell line for bioassay of rhEGF. This cell line, compared with the control cells, had a 2 day shorter operation time and had lower interference. The responsive cell line is more suitable for use in bioassay of rhEGF.