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Cyclin-dependent kinase 2 (CDK2) antagonism inhibits clustering of excessive centrosomes at mitosis, causing multipolar cell division and apoptotic death. This is called anaphase catastrophe. To establish induced anaphase catastrophe as a clinically tractable antineoplastic mechanism, induced anaphase catastrophe was explored in different aneuploid cancers after treatment with CYC065 (Cyclacel), a CDK2/9 inhibitor. Antineoplastic activity was studied in preclinical models. CYC065 treatment augmented anaphase catastrophe in diverse cancers including lymphoma, lung, colon, and pancreatic cancers, despite KRAS oncoprotein expression. Anaphase catastrophe was a broadly active antineoplastic mechanism. Reverse phase protein arrays (RPPAs) revealed that along with known CDK2/9 targets, focal adhesion kinase and Src phosphorylation that regulate metastasis were each repressed by CYC065 treatment. Intriguingly, CYC065 treatment decreased lung cancer metastases in in vivo murine models. CYC065 treatment also significantly reduced the rate of lung cancer growth in syngeneic murine and patient-derived xenograft (PDX) models independent of KRAS oncoprotein expression. Immunohistochemistry analysis of CYC065-treated lung cancer PDX models confirmed repression of proteins highlighted by RPPAs, implicating them as indicators of CYC065 antitumor response. Phospho-histone H3 staining detected anaphase catastrophe in CYC065-treated PDXs. Thus, induced anaphase catastrophe after CYC065 treatment can combat aneuploid cancers despite KRAS oncoprotein expression. These findings should guide future trials of this novel CDK2/9 inhibitor in the cancer clinic.

Fixed-bed bioreactors packed with macrocarriers show great potential to be used for vaccine process development and large-scale production due to distinguishing features of low shear force, high cell adhering surface area, and easy replacement of culture media in situ. As an initial step of utilizing this type of bioreactors for Pseudorabies virus production (PRV) by African green monkey kidney (Vero) cells, we developed a tube-fixed-bed bioreactor in the previous study, which represents a scale-down model for further process optimization. By using this scale-down model, here we evaluated impacts of two strategies (use of serum-free medium and low cell inoculum density) on PRV production, which have benefits of simplifying downstream process and reducing risk of contamination. We first compared Vero cell cultures with different media, bioreactors and inoculum densities, and conclude that cell growth with serum-free medium is comparable to that with serum-containing medium in tube-fixed-bed bioreactor, and low inoculum density supports cell growth only in this bioreactor. Next, we applied serum-free medium and low inoculum cell density for PRV production. By optimization of time of infection (TOI), multiplicity of infection (MOI) and the harvesting strategy, we obtained total amount of virus particles ~ 9 log10 TCID50 at 5 days post-infection (dpi) in the tube-fixed-bed bioreactor. This process was then scaled up by 25-fold to a Xcell 1-L fixed-bed bioreactor, which yields totally virus particles of 10.5 log10 TCID50, corresponding to ~ 3 × 105 doses of vaccine. The process studied in this work holds promise to be developed as a generic platform for the production of vaccines for animal and human health.

To identify the zinc transport function of the membrane proteins Gt1 and Zrt1 in Komagataella phaffii (Pichia pastoris) and study their regulatory mode. Two membrane proteins that might have zinc transport function were found in K. phaffii. GT1 was known to encode a glycerol transporter belonging to the Major Facilitator Superfamily. ZRT1 was predicted to resemble the zinc transporter gene in Saccharomyces cerevisiae. Consistent with the prediction, protein plasma-membrane localizations were confirmed by ultracentrifugation and confocal microscopy. Their zinc binding abilities were identified by ITC in vitro, and the impaired zinc uptake activity caused by their deficiencies was confirmed by zinc fluorescence quantification in vivo. Furthermore, zinc excess could turn the two channels off, while zinc deficiency induced their expressions. Gt1 could only function to maintain zinc homeostasis in glycerol, while the block of Gt1 function might lead to Zrt1 upregulation in glucose. The zinc transport capabilities of Gt1 and Zrt1 were identified in vivo and in vitro. Their regulatory mode to maintain zinc homeostasis in K. phaffii is a new inspiration.

Background Neonatal pneumonia (NP) has a high fatality rate in neonatal illness. This research investigated the functions of emodin on lipopolysaccharide (LPS)-evoked inflammatory injury in WI-38 cells. Methods Cell counting kit-8 (CCK-8) assay and flow cytometry were utilized for examining the impacts of LPS and emodin on viability and apoptosis, respectively. Taurine up-regulated gene 1 (TUG1) level was altered through cell transfection and investigated by reverse transcription quantitative polymerase chain reaction (RT-qPCR). Moreover, RT-qPCR, western blot and enzyme-linked immunosorbent assay (ELISA) were utilized for investigating expressions of monocyte chemoattractant protein-1 (MCP-1) and interleukin (IL)-6. Western blot was carried out for investigating the levels of Bcl-2, Bax, pro-Caspase-3, cleaved-Caspase-3 and NF-κB and p38MAPK pathway-related proteins. Results LPS treatment restrained cell viability, enhanced apoptosis, and expressions of inflammation-related IL-6 and MCP-1. Emodin alleviated LPS-evoked inflammatory injury and restrained the NF-κB and p38MAPK pathways. Furthermore, emodin positively regulated TUG1 expression and TUG1 silencing could reverse the efficacy of emodin on IL-6 and MCP-1 expressions. Finally, TUG1 regulates the expression of inflammatory factors through NF-κB and p38MAPK pathways. Conclusion Emodin alleviated LPS-evoked inflammatory injury by raising TUG1 expression via NF-κB and p38MAPK pathways in WI-38 cells.

Background Infantile pneumonia (IP) is a usual disease in infants and young children. The function and underlying mechanism of circZNF652 on lipopolysaccharide (LPS)-triggered inflammatory damage in WI-38 cells were detected in this article. Methods WI-38 cells were induced by dosages of LPS to construct inflammatory injury model. WI-38 cell viability and apoptosis were detected by CCK-8 assay and flow cytometry, respectively. CircZNF652 and miR-181a levels were changed and detected by cell transfection and qRT-PCR. The levels of apoptosis and JNK/p38 and NF-κB pathways-related proteins, as well as the level of Cox-2 were detected by western blot. Finally, the concentrations of inflammatory factors were detected by ELISA. Results LPS induced inflammatory injury showing as notably decreased the viability, while increased the numbers of apoptotic cells, as well as the levels of apoptosis and inflammatory factors in a dose dependent way. Besides, LPS inducement remarkably enhanced the expression of circZNF652. However, knockdown of circZNF652 remitted LPS-triggered inflammatory damage and restrained NF-κB and JNK/p38 pathways. Moreover, circZNF652 knockdown promoted miR-181a expression. Whereas, miR-181a inhibition markedly relieved circZNF652 knockdown-induced impacts. Conclusion Knockdown of circZNF652 remitted LPS-triggered WI-38 cells inflammatory damage through deactivation of NF-κB and JNK/p38pathways by up-regulating miR-181a.

Supplemental Digital Content is available in the text., Background: Human loss-of-function variants of ANK2 (ankyrin-B) are linked to arrhythmias and sudden cardiac death. However, their in vivo effects and specific arrhythmogenic pathways have not been fully elucidated. Methods: We identified new ANK2 variants in 25 unrelated Han Chinese probands with ventricular tachycardia by whole-exome sequencing. The potential pathogenic variants were validated by Sanger sequencing. We performed functional and mechanistic experiments in ankyrin-B knockin (KI) mouse models and in single myocytes isolated from KI hearts. Results: We detected a rare, heterozygous ANK2 variant (p.Q1283H) in a proband with recurrent ventricular tachycardia. This variant was localized to the ZU5C region of ANK2, where no variants have been previously reported. KI mice harboring the p.Q1283H variant exhibited an increased predisposition to ventricular arrhythmias after catecholaminergic stress in the absence of cardiac structural abnormalities. Functional studies illustrated an increased frequency of delayed afterdepolarizations and Ca2+ waves and sparks accompanied by decreased sarcoplasmic reticulum Ca2+ content in KI cardiomyocytes on isoproterenol stimulation. The immunoblotting results showed increased levels of phosphorylated ryanodine receptor Ser2814 in the KI hearts, which was further amplified on isoproterenol stimulation. Coimmunoprecipitation experiments demonstrated dissociation of protein phosphatase 2A from ryanodine receptor in the KI hearts, which was accompanied by a decreased binding of ankyrin-B to protein phosphatase 2A regulatory subunit B56α. Finally, the administration of metoprolol or flecainide decreased the incidence of stress-induced ventricular arrhythmias in the KI mice. Conclusions: ANK2 p.Q1283H is a disease-associated variant that confers susceptibility to stress-induced arrhythmias, which may be prevented by the administration of metoprolol or flecainide. This variant is associated with the loss of protein phosphatase 2A activity, increased phosphorylation of ryanodine receptor, exaggerated delayed afterdepolarization-mediated trigger activity, and arrhythmogenesis.

Ubiquitin specific peptidase 18 (USP18), previously known as UBP43, is the IFN-stimulated gene 15 (ISG15) deconjugase. USP18 removes ISG15 from substrate proteins. This study reports that USP18-null mice (vs. wild-type mice) exhibited lower lipolysis rates, altered fat to body weight ratios, and cold sensitivity. USP18 is a regulator of lipid and fatty acid metabolism. Prior work established that USP18 promotes lung tumorigenesis. We sought to learn whether this occurs through altered lipid and fatty acid metabolism. Loss of USP18 repressed adipose triglyceride lipase (ATGL) expression; gain of USP18 expression upregulated ATGL in lung cancer cells. The E1-like ubiquitin activating enzyme promoted ISG15 conjugation of ATGL and destabilization. Immunoprecipitation assays confirmed that ISG15 covalently conjugates to ATGL. Protein expression of thermogenic regulators was examined in brown fat of USP18-null versus wild-type mice. Uncoupling protein 1 (UCP1) was repressed in USP18-null fat. Gain of USP18 expression augmented UCP1 protein via reduced ubiquitination. Gain of UCP1 expression in lung cancer cell lines enhanced cellular proliferation. UCP1 knockdown inhibited proliferation. Beta-hydroxybutyrate colorimetric assays performed after gain of UCP1 expression revealed increased cellular fatty acid beta-oxidation, augmenting fatty acid beta-oxidation in Seahorse assays. Combined USP18, ATGL, and UCP1 profiles were interrogated in The Cancer Genome Atlas. Intriguingly, lung cancers with increased USP18, ATGL, and UCP1 expression had an unfavorable survival. These findings reveal that USP18 is a pharmacologic target that controls fatty acid metabolism. Implications: USP18 is an antineoplastic target that affects lung cancer fatty acid metabolism.

Pichia pastoris is able to metabolize methanol via a specific MUT (methanol utilization) pathway. Based on the powerful AOX1 (Alcohol Oxidase 1) promoter, the P. pastoris expression system has become one of the most widely used eukaryotic expression systems. The molecular mechanisms of methanol metabolic regulation remain unclearly understood, so it is important to identify and develop new transcriptional regulators. Our previous studies suggested that the expression of SUT2 could be induced by methanol but is repressed by glycerol, which indicates that SUT2 may be involved in methanol metabolism through an unknown mechanism. SUT2 encodes a putative transcription factor-like protein harboring a Gal4-like Zn2Cys6 DNA-binding domain in Pichia pastoris, and its homolog in Saccharomyces cerevisiae regulates sterol uptake and synthesis. This study shows that the overexpression of SUT2 promoted the expression of AOX1 and increases ergosterol content in cells. Furthermore, via truncation of the putative SUT2 promoter at diverse loci, the − 973 base pair (bp) to − 547 bp region to the ATG was shown to be the core element of the inducible promoter PSUT2, which strongly responds to the methanol signal. The transcriptional start site of SUT2, “A” at the 22nd bp upstream of ATG, was determined with 5′-rapid amplification of cDNA ends. A forward-loop cassette was constructed with MXR1 (Methanol Expression Regulator 1, a positive transcription factor of PAOX1) promoted by PSUT2, enabling moderate elevation in the expression level of Mxr1 and high activity of PAOX1 without damaging cellular robustness further boosting the production of heterologous proteins. The PAOX1-driven expression of enhanced green fluorescent protein in this novel system was improved by 18%, representing a promising method for extrinsic protein production. SUT2 may play roles in methanol metabolism by participating in sterol biosynthesis. PSUT2 was characterized as a novel inducible promoter in P. pastoris and a PSUT2-driven MXR1 forward-loop cassette was constructed to enhance the PAOX1 activity, laying a foundation for further development and application of P. pastoris expression system.

The dissolved oxygen (DO) level of a culture of Corynebacterium glutamicum (C. glutamicum) in a bioreactor has a significant impact on the cellular redox potential and the distribution of energy and metabolites. In this study, to gain a deeper understanding of the effects of DO on the metabolism of C. glutamicum, we sought to systematically explore the influence of different DO concentrations on genetic regulation and metabolism through transcriptomic analysis. The results revealed that after 20 h of fermentation, oxygen limitation enhanced the glucose metabolism, pyruvate metabolism and carbon overflow, and restricted NAD+ availability. A high oxygen supply enhanced the TCA cycle and reduced glyoxylate metabolism. Several key genes involved in response of C. glutamicum to different oxygen concentrations were examined, which provided suggestions for target site modifications in developing optimized oxygen supply strategies. These data provided new insights into the relationship between oxygen supply and metabolism of C. glutamicum.

Background Bronchopneumonia is a common lower respiratory tract inflammatory disease, which is one of main causes of death in infants and young children. Tanshinone IIA (Tan) is a naturally derived anti-inflammatory compound, which has been used for some inflammatory diseases. Objective Tan was expected to inhibit lipopolysaccharide (LPS)-induced inflammatory damage in human bronchial epithelium cell line BEAS-2B. Methods BEAS-2B cells were stimulated with LPS to mimic an in vitro model of bronchopneumonia. Cell viability and apoptosis were respectively assessed by CCK-8 assay and flow cytometry. The pro-inflammatory cytokines (IL-1β, IL-6 and TNF-α) were analyzed by qRT-PCR and quantified by ELISA. Relative miR-27a expression and Bcl-2 expression were detected by qRT-PCR. Cyclin D1 expression, Bcl-2 expression, and main factors in PI3K/AKT and JNK signaling pathways were analyzed by Western blotting. Dual luciferase activity assay was conducted to analyze the target gene of miR-27a. Results Treatment of LPS inhibited cell viability, suppressed expression of Cyclin D1, promoted apoptosis, and enhanced secretions of IL-1β, IL-6 and TNF-α, whereas, all effects were reversed by Tan treatment. Moreover, miR-27a expression was decreased by Tan. Tan protected BEAS-2B cells by down-regulating miR-27a. Bcl-2 was a direct target of miR-27a and Bcl-2 overexpression attenuated the cell injury-promoting effect of miR-27a. PI3K/AKT and JNK signaling pathways were inhibited via action of Tan-miR-27a axis. Conclusion Overall, Tan could protect BEAS-2B cells against LPS-induced inflammation damage via inactivating PI3K/AKT and JNK pathways by down-regulating miR-27a.

Algicidal bacteria associated with Karenia mikimotoi have been isolated, yet the distribution of the algicidal bacteria has been rarely studied. Here, we postulated and demonstrated that terrestrial environment harbors diverse algicidal bacteria, which can survive in seawater along water flowing into marine and suppress Karenia mikimotoi. In summary, 9 and 5 bacteria with algicidal activity on Karenia mikimotoi were isolated from seawater and estuarine soil, respectively. Similar with the marine bacteria (Alteromonas sp., Halomonas sp., Marinobacter sp., Paracoccus sp., Rhodobacteraceae, Idiomarina sp.), the soil strains (Pseudoalteromonas sp. and Flavobaterium sp.) showed high mortality in Karenia mikimotoi with the inhibitory rate of 87% and 93.5%, respectively, after two days co-cultivation. Algicidal activity of the two strains was detected in the cell-free filtrate not in bacterial cells. The results suggest that algicidal bacteria associated with Karenia mikimotoi widely exist in terrestrial and marine environments, and have application potential on controlling Karenia mikimotoi.

c-Jun N-terminal kinase (JNK) contributes to the pathogenesis of diabetic nephropathy (DN). The JNK inhibitor SP600125 was reported to ameliorate DN. However, the mechanism remained unclear. We previously reported that SP600125 activated nuclear factor erythroid 2-related factor 2 (NRF2), a governor of the cellular antioxidant defense system, in the aortas of the diabetic mice. Given the critical role of NRF2 in preventing DN, the present study aimed to test whether or not NRF2 is required for SP600125’s protection against DN. To test the role of NRF2 in SP600125’s effect, streptozotocin-induced C57BL/6 wild-type (WT) andNrf2-knockout (KO) diabetic mice were treated in the presence or absence of SP600125, for 24 weeks. To explore the mechanism by which SP600125 activates NRF2, mouse mesangial cells (MMCs) were treated with high glucose (HG), in the presence or absence of either SP600125 or JNK siRNA. SP600125 significantly attenuated the diabetes-induced renal oxidative stress, inflammation, fibrosis, pathological change and dysfunction in the WT, but not theNrf2KO mice. SP600125 inactivated JNK, inhibited kelch-like ECH-associated protein 1 expression, preserved NRF2 protein and facilitated its nuclear translocation in the kidneys of the WT mice, the effects of which were similarly produced by either SP600125 or JNK siRNA in HG-treated MMCs. Further, both SP600125 and JNK siRNA alleviated HG-induced mesangial oxidative stress and expression of inflammatory and fibrotic genes. The present study demonstrates that NRF2 is required for SP600125’s protection against DN. SP600125 activates NRF2 possibly via inhibition of JNK-inducedKeap1expression.

Hepatocellular carcinoma (HCC) is a common cancer worldwide with an aggressive and highly proliferative activity. Studies had confirmed that HCC cell proliferation is associated with the cell cycle's G1 phase, but the detailed molecular mechanism has not been thoroughly elucidated to date. Eukaryotic translation elongation factor 1A1 (eEF1A1) is an evolutionarily conserved elongation factor protein and is involved in tumor cell proliferation. However, which phase of the cell cycle is regulated by eEF1A1 to influence cell proliferation in HCC and its detailed molecular mechanism remain unclear. In this study, we observed that eEF1A1 influences HCC cell proliferation by regulating the cell cycle's G1 phase. In addition, eEF1A1 influences G1 phase by regulating cyclin D1 expression, promoting HCC cell proliferation both in vitro and in vivo. Moreover, our results indicated that eEF1A1 regulates cyclin D1 expression through STAT1 signaling. STAT1 increases the transcriptional activity of cyclin D1 by binding to the cyclin D1 promoter. Taken together, these findings enabled us to identify a novel mechanism by which eEF1A1 regulates the cell cycle's G1 phase to promote tumor proliferation by regulating cyclin D1 expression through STAT1 signaling in HCC.

Corynebacterium glutamicum is an attractive host for the production of heterologous proteins despite its traditional use in fermentative production of amino acids. To enhance the expression levels of target genes, the development of useful promoters is required in the construction of expression systems. Here, we developed a new promoter, the aph promoter from aminoglycoside-3'-phosphotransferase gene, and used it to construct monocistronic and bicistronic expression systems that host different ribosome binding site (RBS) sequences. First, the expression level of the reporter protein, enhanced green fluorescent protein (EGFP), varied with changes in the RBS sequences in the constructed vectors. The results showed that the fluorescence intensities of the bicistronic group were higher than those of the monocistronic group and that RM3E showed the highest fluorescence intensity, which was 42-fold higher than the lowest (RA2E') among these groups. Next, taking advantage of the optimized aph promoter, we successfully employed this aph promoter for α-amylase and VHH (camelid antibody fragment) expression. The secretion of α-amylase improved 1.5-fold after promoter mutation. This promoter will be useful for heterologous protein production in C. glutamicum cells.

The dissolved oxygen (DO) level of a culture of Corynebacterium glutamicum (C. glutamicum) in a bioreactor has a significant impact on the cellular redox potential and the distribution of energy and metabolites. In this study, to gain a deeper understanding of the effects of DO on the metabolism of C. glutamicum, we sought to systematically explore the influence of different DO concentrations on genetic regulation and metabolism through transcriptomic analysis. The results revealed that after 20 h of fermentation, oxygen limitation enhanced the glucose metabolism, pyruvate metabolism and carbon overflow, and restricted NAD+ availability. A high oxygen supply enhanced the TCA cycle and reduced glyoxylate metabolism. Several key genes involved in response of C. glutamicum to different oxygen concentrations were examined, which provided suggestions for target site modifications in developing optimized oxygen supply strategies. These data provided new insights into the relationship between oxygen supply and metabolism of C. glutamicum.

Oxidative stress contributes to the pathogenesis of diabetic nephropathy (DN). Nuclear factor erythroid 2-related factor 2 (NRF2) plays a key role in cellular defense against oxidative stress. NRF2 activators have shown promising preventive effects on DN. Sodium butyrate (NaB) is a known activator of NRF2. However, it is unknown whether NRF2 is required for NaB protection against DN. Therefore, streptozotocin-induced diabetic C57BL/6 Nrf2 knockout and their wild-type mice were treated in the presence or absence of NaB for 20 weeks. Diabetic mice, but not NaB-treated diabetic mice, developed significant renal oxidative damage, inflammation, apoptosis, fibrosis, pathological changes and albuminuria. NaB inhibited histone deacetylase (HDAC) activity and elevated the expression of Nrf2 and its downstream targets heme oxygenase 1 and NAD(P)H dehydrogenase quinone 1. Notably, deletion of the Nrf2 gene completely abolished NaB activation of NRF2 signaling and protection against diabetes-induced renal injury. Interestingly, the expression of Kelch-like ECH-associated protein 1, the negative regulator of NRF2, was not altered by NaB under both diabetic and non-diabetic conditions. Moreover, NRF2 nuclear translocation was not promoted by NaB. Therefore, the present study indicates, for the first time, that NRF2 plays a key role in NaB protection against DN. Other findings suggest that NaB may activate Nrf2 at the transcriptional level, possibly by the inhibition of HDAC activity.

DTNA encoding dystrobrevin-α (α-DB) is a putative causal gene associated with left ventricular noncompaction cardiomyopathy (LVNC). The aim of the study was to investigate the causal role of DTNA in LVNC using a transgenic mouse model.A missense mutation (c.146A > G, p.N49S) of DTNA was identified in a patient with LVNC by Sanger sequencing. Six independent lines of transgenic mice expressing the mutant DTNA under a myosin heavy chain 6 (Myh6) promoter were generated (Myh6:DtnaN49S). Phenotypic characteristics of DTNA-p.N49S mutations were evaluated by echocardiography, histological observation, and immunoblotting. Multiple trabeculation and a higher ratio of non-compacted to compact myocardial layer were found in the Myh6:DtnaN49S mice compared to the controls. The transgenic mice also showed left ventricular (LV) dilation and cardiac systolic dysfunction. In conclusion, overexpression of the DTNA-p.N49S mutation in a mouse heart can be responsible for the phenotype of deep trabeculation, dilated cardiomyopathy, and cardiac dysfunction, which resembles the phenotype of LVNC.

This study described a successful application of the Quality by Design (QbD) approach to pseudorabies virus (PRV) production process development in a fixed-bed bioreactor using the serum-free medium (SFM). The innovated tube-fixed-bed bioreactor was used as a scale-down model of the fixed-bed bioreactor for process development. Risk analysis was performed using Ishikawa diagram combined with failure mode effects analysis (FMEA). The comparative experiment was performed to screen proper medium for adherent African green monkey kidney (Vero) cells from three commercially available SFMs (VP-SFM, ProVERO-1 and Vero-A). The Vero-A medium showed as an outstanding one for further study. The PRV titer in harvest medium was consider as Critical Quality Attribute (CQA) and the Critical Process Parameters (CPPs) [time of infection (TOI), multiplicity of infection (MOI) and initial inoculation cell density] ranked high with risk priority number (RPN) were taken into design of experiment (DoE) methodology. Then prediction model of PRV production process was established and a robust PRV production process was explored. Under the robust setpoint conditions, the Xcell 1 L laboratory-scale fixed-bed bioreactor yielded PRV titer up to 7.87 log(10) TCID(50)/mL at 3 dpi, which was comparable with that in the tube-fixed-bed bioreactor. Combination of the tube-fixed-bed bioreactor and QbD approach could further accelerate the development of a robust virus production process.

Transforming growth factor-β-activated kinase 1 (TAK1)-binding protein 3 (TAB3) and the proviral integration site for Moloney murine leukaemia virus 1 (PIM1) are implicated in cancer development. In this study, we investigated the relationship between TAB3 and PIM1 in colorectal cancer (CRC) and determined the potential role and molecular mechanism of TAB3 in PIM1-mediated CRC growth. We found that TAB3 and PIM1 expression levels were positively correlated in CRC tissues. The knockdown of TAB3 significantly decreased PIM1 expression and inhibited CRC proliferation in vitro and in vivo. The upregulation of PIM1 rescued the decreased cell proliferation induced by TAB3 knockdown, whereas PIM1 knockdown decreased TAB3-enhanced CRC proliferation. Additionally, TAB3 regulates PIM1 expression through the STAT3 signalling pathway and confirmed a positive correlation between TAB3 and phosphorylated-STAT3 expression in CRC tissues. Patients with high expression of TAB3 and phosphorylated-STAT3 had the worst prognosis. Mechanistically, TAB3 regulates PIM1 expression by promoting STAT3 phosphorylation and activation through the formation of the TAB3-TAK1-STAT3 complex. Overall, a novel CRC regulatory circuit involving the TAB3-TAK1-STAT3 complex and PIM1 was identified, the dysfunction of which may contribute to CRC tumorigenesis.

Corynebacterium glutamicum is an important industrial organism for the production of a variety of biological commodities. We discovered a promoter encoded by the gene NCgl2319 in C. glutamicum, which could be induced by benzyl alcohol, could be used as an efficient tunable expression system. In initial attempts, this promoter failed to function in a recombinant expression system. This was remedied by extending the original genetic context of the promoter, generating a new version Pcat-B. The Pcat-B transcription initiation site, its critical active regions, and its effect of inducers were fully characterized resulting in tunable expression. This approach proved to be very efficient in producing a pharmaceutical protein, N-terminal pro-brain natriuretic peptide (NT-proBNP). Production of approximately 440.43 mg/L NT-proBNP was achieved with the Pcat-B expression system demonstrating its application for controllable pharmaceutical protein production in C. glutamicum.

Cell lysis has long been a process problem in protein expression using bacterial hosts. To explore the pathways involved in cell lysis in a bioreactor, Escherichia coli strain W3110 was used as the host to express single-chain variable-domain antibody fragments (scFv). Under the same fermentation conditions, E. coli strain W3110 expressing a humanized scFv entered the viable but nonculturable (VBNC) state 6 h before the wild-type strain, and the accumulation of the scFv within the cells accelerated cell lysis. At the transcriptional level, the scFv not only altered the expression of rpoH, dnaK, dnaJ, groES, and groEL in the heat stress response, but also rpoS, gadE, and gadX in the acid stress response pathway and sodA and katE in the oxygen stress response pathway. During cell lysis in a fermenter, the expression of the membrane protein-encoding genes ompA, ompC, ompW, and ompX significantly decreased, and the high-level expression of rpoE was not sustained. These findings provide new insights into cell lysis as well as a theoretical foundation for improving fermentation conditions and engineering bacteria to minimize cell lysis during fermentation.

Pichia pastoris is a methylotrophic yeast in which host heterologous expression of proteins has been developed owing to the strong inducible alcohol oxidase promoter (PAOX1). However, it is difficult to manipulate the genome in P. pastoris. Based on previous attempts to apply the CRISPR/Cas9 system in P. pastoris, a CRISPR/Cas9 system with episomal sgRNA plasmid was developed and 100 % genome editing efficiency, high multicopy gene editing and stable multigene editing were obtained without a sharp decline caused by multi-sgRNA. And 28/34 (∼82 %) sgRNAs tested were effective. The CGG may have a slightly higher and more stable cleavage efficiency than the other three NGG motifs, and a low GC content may be preferable for higher cleavage efficiency. This provides researchers with a stable genome editing tool that shows a high editing efficiency, shortening the experimentation period. Furthermore, we introduced dCas9 into P. pastoris and achieved target gene interference, expanding the CRISPR/Cas9 toolbox in P. pastoris.

Glucose-regulated protein 78(GRP78) and the ubiquitin-like protein FAT10 each promote proliferation in hepatocellular carcinoma(HCC). However, the relationship of GRP78 and FAT10 in HCC proliferation are still not known. In this study, we found that GRP78 and FAT10 were significantly overexpressed in HCC tissues compare with adjacent non-cancerous tissues, and a positive correlation was found between their expression and associated proliferation characteristics. High expression of GRP78 and FAT10 were positively correlated with tumor proliferation and poor prognosis in HCC. Moreover, GRP78 knockdown reduced FAT10 expression and suppressed HCC proliferation in vitro and in vivo. The effects of GRP78 knockdown were rescued by FAT10 up-regulation, whereas FAT10 knockdown reduced HCC proliferation enhanced by GRP78 up-regulation. Furthermore, GRP78 modulated FAT10 expression by regulating the NF-κB pathway, direct activation of the NF-κB pathway increased the expression of FAT10, a gene counteracting the tumor suppressor p53. Taken together, these results suggest that this newly identified GRP78–NF-κB–FAT10 axis will provide novel insight into the understanding of the regulatory mechanisms of proliferation in human HCC.

Recently, studies on transcriptome-proteome relationships have revealed mRNA/protein expression discordance for certain genes and speculated that protein posttranslational modification (PTM) may be involved. However, there is currently no evidence to support this hypothesis. Wnt-induced secreted protein-1 (WISP1) is the downstream target gene of β-catenin and plays an important role in tumorigenesis and progression, but the expression and role of WISP1 in different tumor types are controversial. Here, we first confirmed that WISP1 protein expression was significantly down-regulated in hepatocellular carcinoma (HCC) tissue and could be an independent predictor of poor prognosis for patients with HCC. In vivo and in vitro evidence was provided that WISP1 can suppress HCC cell proliferation. Further studies have found that low WISP1 protein expression was related to expression of human leukocyte antigen F locus adjacent transcript 10 (FAT10), a specific ubiquitin-like protein with both degradation and stabilization functions, which plays an important role in PTM. FAT10 overexpression facilitated WISP1 degradation by FAT10ylation to decrease WISP1 protein expression, thus promoting HCC proliferation. Interestingly, we found and demonstrated that FAT10 overexpression could result in WISP1 protein/mRNA expression discordance, with protein expression decreasing while mRNA expression increased. The underlying mechanism is that FAT10 exerts substrate stabilization and degradation functions simultaneously, while FAT10 overexpression promotes WISP1 mRNA expression by stabilizing β-catenin and directly degrades WISP1 protein. Conclusion: Our study demonstrated that overexpression of FAT10 results in expression discordance between WISP1 protein and mRNA, thereby promoting HCC progression by down-regulating WISP1 protein expression.

Real-time quantitative PCR (qRT-PCR) is a fast and efficient technology for detecting gene expression levels in the study of the Corynebacterium glutamicum protein expression system, but it requires normalization to ensure the reliability of the results obtained. We selected 13 genes from the commonly used housekeeping genes and from transcriptome data as candidate reference genes. The Ct values of the 13 genes were obtained by qRT-PCR at different fermentation stages and under three stress conditions (temperature, acid and salt). The expression stability of the reference genes was evaluated by geNorm and NormFinder software. For the study of different growth stages, the most appropriate reference genes are Ncgl2772 and leua, which encode acetyl-CoA carboxylase beta subunit and 2-isopropylmalate synthase, separately. For the study of different stress factors, the optimal minimum number of reference genes is 3, with Ncgl2772, gyrb encoding DNA gyrase B and siga encoding RNA polymerase sigma factor A as the most suitable combination. Additionally, clpx and clpc, encoding ClpX and ClpC protease subunits, were used to validate the candidate reference genes. The identification of new reference genes makes qRT-PCR more convenient, and using these genes for normalization can improve the accuracy and reliability of the measurements of target gene expression levels obtained by qRT-PCR for C. glutamicum.

Bronchopneumonia is a common multiple infection disease under 2 years old. Luteolin is a natural flavonoid widely distributed in plants with anti-inflammatory effect. This study aimed to explore the effects of luteolin on lipopolysaccharide (LPS)-induced bronchopneumonia injury in vitro and in vivo. Firstly, the viability and apoptosis of human bronchial epithelial BEAS-2B cells after luteolin treatment were assessed. Then, cells were treated with 10 μM LPS to simulate inflammatory injury. The potential protective effects of luteolin on LPS-induced BEAS-2B cell inflammatory injury were detected. Moreover, after LPS and/or luteolin treatment, the expression of microRNA-132 (miR-132) in BEAS-2B cells was measured. The roles of miR-132 in protective activity of luteolin were investigated. Finally, the LPS-induced bronchopneumonia murine model was established and the anti-inflammatory effects of luteolin in vivo were analyzed. The results showed that LPS decreased BEAS-2B cell viability, increased cell apoptosis and enhanced inflammatory cytokines expression. Luteolin alleviated the LPS-induced viability loss, apoptosis and elevated expression of inflammatory cytokines in a dose-dependent manner. Moreover, luteolin alleviated the LPS-induced miR-132 expression increase in BEAS-2B cells. Overexpression of miR-132 reversed the protective effects of luteolin on LPS-induced inflammatory injury. Mechanistically, luteolin mitigated LPS-induced activation of NF-κB signaling pathway by down-regulation of miR-132. Furthermore, we also found that luteolin alleviated LPS-induced bronchopneumonia model in vivo. In conclusion, this study revealed that luteolin alleviated LPS-induced bronchopneumonia injury in vitro and in vivo through down-regulating miR-132. These findings provide theoretical basis for deeply exploring the treatment of bronchopneumonia in children by using luteolin.

Background Corynebacterium glutamicum (C. glutamicum) has traditionally been used as a microbial cell factory for the industrial production of many amino acids and other industrially important commodities. C. glutamicum has recently been established as a host for recombinant protein expression; however, some intrinsic disadvantages could be improved by genetic modification. Gene editing techniques, such as deletion, insertion, or replacement, are important tools for modifying chromosomes. Results In this research, we report a CRISPR/Cas9 system in C. glutamicum for rapid and efficient genome editing, including gene deletion and insertion. The system consists of two plasmids: one containing a target-specific guide RNA and a homologous sequence to a target gene, the other expressing Cas9 protein. With high efficiency (up to 100%), this system was used to disrupt the porB, mepA, clpX and Ncgl0911 genes, which affect the ability to express proteins. The porB- and mepA-deletion strains had enhanced expression of green fluorescent protein, compared with the wild-type stain. This system can also be used to engineer point mutations and gene insertions. Conclusions In this study, we adapted the CRISPR/Cas9 system from S. pyogens to gene deletion, point mutations and insertion in C. glutamicum. Compared with published genome modification methods, methods based on the CRISPR/Cas9 system can rapidly and efficiently achieve genome editing. Our research provides a powerful tool for facilitating the study of gene function, metabolic pathways, and enhanced productivity in C. glutamicum. Electronic supplementary material The online version of this article (10.1186/s12934-017-0814-6) contains supplementary material, which is available to authorized users.

Directly using the promoter associated with 5'-untranslated region of a high-protein-abundance gene from the genome may cause low expression activity of an expression system. A bicistronic expression part containing the short 5' coding sequence of the source gene and an embedded Shine-Dalgarno sequence can cause higher expression levels of the recombinant gene in a bicistronic cassette. Here, we evaluated two methods to construct expression parts and exploited genomic sequence sources to provide specific functional sequences to complete the expression system. The architecture of the bicistronic part increased the expression levels of target genes and performed more reliably than conventional expression parts with the same promoter and 5' untranslated region. For Corynebacterium glutamicum, the strongest bicistronic part, HP-BEP4, was obtained from a heterologous sequence source, leading to a 2.24-fold increase in the expression level of fluorescent protein over constitutively expressed pXMJ19 or the production of more than 100 mg/L single-chain variable fragment (scFv). It could meet the needs of overexpressing key genes in C. glutamicum.

The efficient promoter of alcohol oxidase 1 (PAOX1) in methylotrophic yeast Pichia pastoris is strictly induced by methanol but repressed by glycerol with an unclear molecular mechanism. In the present study, the gene of a previously characterized transmembrane protein glycerol transporter 1 (GT1) of P. pastoris GS115 was deleted by homologous recombination. Transcriptional profiles of the mutant (gt1Δ) and wild type (WT) were compared with different carbon sources (glycerol, methanol and glycerol-methanol mix) at various time points using high-throughput RNA-Seq techniques. We determined that the loss of glycerol transporter 1 (Gt1p) could relieve catabolite repression in the glycerol-methanol mixed medium and shared a similar transcriptional profile with the WT in methanol medium. By calculating the common differentially expressed genes in three distinct paired groups, genes involved in the stress response, nutrition deprivation and translational process were identified, explaining the potential roles of glycerol in the regulation of methanol metabolism. Based on weighted gene co-expression network analysis, the relationship between biological traits and the transcriptional profile was established. With the support of published research and our data, we propose two possible regulatory pathways that are involved in the regulation of catabolite repression (adenosine 5΄-monophosphate (AMP)-activated protein kinase /SNF1 and Mitogen-activated protein kinase/HOG), thereby providing potential targets for both research and industrial strain improvement.

// Chuqian Zheng 1, 2, 3 , Xiuxia Liu 2 , Leifeng Chen 1, 2, 3 , Zheng Xu 1, 2, 3 and Jianghua Shao 1, 2, 3 1 Department of General Surgery, Second Affiliated Hospital of Nanchang University, Nanchang, 330000, China 2 Jiangxi Province Key Laboratory of Molecular Medicine, Nanchang, 330000, China 3 Jiangxi Province Engineering Research Center of Hepatobiliary Disease, Nanchang, 330000, China Correspondence to: Jianghua Shao, email: shao5022@163.com Keywords: lncRNA, hepatocellular carcinoma (HCC), survival, prognostic, meta-analysis Received: April 07, 2017 Accepted: July 12, 2017 Published: July 25, 2017 ABSTRACT The latest studies have shown that long non-coding RNAs (lncRNAs) may be considered markers as their expression levels were abnormal in cancer and can be used as a molecular biomarker for the potential assessment of cancer prognosis. In this study, we aimed to assess the prognostic value of lncRNA as marker of patients with hepatocellular carcinoma. We performed a detailed search of the PubMed and Embase databases for articles on the prognostic value of various lncRNAs in HCC. We then carefully extracted the relevant data from the articles, and we used the meta-analysis method to analyze these results; heterogeneity and publication bias were also evaluated. With 40 associative studies included, we found that high expression of 27 types of lncRNA was associated with a poor prognosis in HCC patients, and low expression of 18 types of lncRNAs was associated with a worse prognosis. Patients with higher lncRNA expression had significantly poor overall survival (OS; pooled HR, 1.25; 95% confidence interval [CI], 1.03–1.52) as well as significantly poor recurrence-free survival (RFS; pooled HR, 1.66; 95% CI, 1.26–2.17). Overexpression of lncRNAs may not meaningfully predict disease-free survival (DFS; pooled HR, 1.04; 95% CI, 0.52–2.07; p = 0.91). Our meta-analysis demonstrated that lncRNAs may serve as predictive biomarkers for cancer prognosis.

In the methylotrophic yeast Pichia pastoris (P. pastoris), the efficient promoter of alcohol oxidase (PAox1) is induced by methanol and repressed by glycerol, but the molecular mechanism is not clear. In this study, the relationship between alcohol oxidase 1 (aox1), methanol expression regulator 1 (mxr1) and glycerol transporter 1 (gt1) was studied. By RT-PCR, it was found that the overexpression of gt1 could increase the glycerol content in cells and repress the expression of mxr1 and aox1, and the deletion of gt1 reduced the glycerol content in cells and promoted the expression of aox1. The overexpression of mxr1 could repress the expression of gt1, and the deletion of mxr1 could promote the expression of gt1 to some extent. By EMSA, Mxr1 binding sites were found in the promoter of gt1 (PGt1) (-141 to -138, CCCC), and Mxr1 could regulate the expression of gt1 by binding to PGt1. The relationships among aox1, mxr1 and gt1 revealed here provide a reference for the understanding of the mechanism of glycerol repression of PAox1.

Epigallocatechin gallate (EGCG) is the most abundant and effective green tea catechin and has been reported to attenuate diabetic nephropathy (DN). However, the mechanism by which EGCG ameliorates DN, till now, has remained unclear. EGCG is known as a potent activator of nuclear factor erythroid 2-related factor 2 (NRF2), which plays a key role in cellular defense against diabetes-induced oxidative stress and in the prevention of DN. In the present study, we tested whether NRF2 is required for EGCG protection against DN. Therefore, C57BL/6 wild type (WT) and Nrf2 knockout mice were induced to diabetes by streptozotocin, in the presence or absence of a 24-week treatment with EGCG. In the WT mice, EGCG activated Nrf2 expression and function without altering the expression of Kelch-like ECH-associated protein 1 (Keap1). Diabetes-induced renal oxidative damage, inflammation, fibrosis and albuminuria were significantly prevented by EGCG. Notably, deletion of the Nrf2 gene completely abrogated these actions of EGCG. To further determine the effect of EGCG on KEAP1/NRF2 signaling, mouse mesangial cells were treated with high glucose, in the presence of both Keap1 siRNA and EGCG. Interestingly, EGCG failed to enhance NRF2 signaling and alleviate oxidative, inflammatory and fibrotic indicators, in the presence of Keap1 siRNA. The present study demonstrated, for the first time, that NRF2 plays a critical role in EGCG protection against DN. Other findings indicated that inactivation of KEAP1 protein by EGCG may mediate EGCG function in activating NRF2.

Rho-associated coiled-coil forming protein kinase 2 (Rock2), as a key effector of the small GTPase RhoA, is involved in tumor development. Scavenger receptor class A member 5 (SCARA5) is an important regulator of biological processes in cancer cells. However, the roles and relationship of Rock2 and SCARA5 in renal cell carcinoma (RCC) remain unclear. In this study, we found that Rock2 expression was markedly increased in clinical RCC tissues compared with that in adjacent non-cancerous tissues. High expression of Rock2 was inversely correlated with patient survival in RCC, which indicated that Rock2 may be a prognostic marker in human RCC. In addition, Rock2 knockdown increased SCARA5 expression and suppressed RCC cell proliferation both in vitro and in vivo. Furthermore, we found that the β-catenin/TCF4 pathway contributed to the effect of Rock2 on SCARA5-mediated RCC proliferation. Taken together, these results suggest that this newly identified Rock2-β-catenin/TCF4-SCARA5 axis will provide novel insight into the understanding of the regulatory mechanisms of proliferation in human RCC.

To construct effective genetic expression parts controlling transcription and translation initiation for synthetic biology and heterologous expression in Corynebacterium glutamicum. Twelve highly expressed genes were identified from the proteomic data of C. glutamicum. Their related sequences were used to construct bicistronic genetic expression parts. Each part contain promoter, 5′-UTR, N-terminal sequence of the source gene and a conserved SD sequence, associated with target gene, forming the bicistronic expression cassette. The enhanced green fluorescent protein (EGFP) expression levels controlled by these novel parts have 1.4 to 790-fold increase in C. glutamicum compared with corresponding promoter-5′-UTR part. One of the bicistronic parts is 1.35 times the EGFP expression of the constitutive-expression pXMJ19. These bicistronic parts had expression advantage compared with conventional promoter-5′-UTR parts. Various genetic parts for efficient gene expression can be quickly obtained via this new method.

Maize is a globally important crop, and a low phosphate (LP) supply frequently limits maize yields in many areas. microRNAs (miRNAs) play important roles in plant development and environmental adaptation. In this study, spatio-temporal miRNA transcript profiling and some of the target genes in the roots and leaves of the maize inbred line Q319 were analyzed in response to LP. Complex small RNA populations were detected after LP culture, and they displayed different patterns in the roots and leaves. Differentially expressed miRNAs can be grouped into 'early' miRNAs, which respond rapidly and are often non-specific to phosphate deficiency, and 'late' miRNAs, which alter the morphology, physiology or metabolism of plants upon prolonged phosphate deficiency. miR827 and miR399-mediated posttranscriptional pathway responses to phosphate availability were conserved and species-specific in maize. Abiotic stress-related miRNAs were engaged in interactions with different signaling and/or metabolic pathways. Auxin-related miRNAs and their targets' expression may be involved in root architecture modification and upland growth retardation in maize when subjected to LP. The changes that were found in the expression of miRNAs and their target genes suggested that miRNA regulation/alterations are pivotal mechanisms in maize adaptations to LP environments. A complex regulatory mechanism involving miRNAs in response to the LP environment is present in maize.

Promoter of alcohol oxidase I (PAOX1) is the most efficient promoter involved in the regulation of recombinant protein expression in Pichia pastoris (P. pastoris). PAOX1 is tightly repressed by the presence of glycerol in the culture medium; thus, glycerol must be exhausted before methanol can be taken up by P. pastoris and the expression of the heterologous protein can be induced. In this study, a candidate glycerol transporter (GT1, GeneID: 8197545) was identified, and its role was confirmed by further studies (e.g. bioinformatics analysis, heterologous complementation in Schizosaccharomyces pombe (S. pombe)). When GT1 is co-expressed with enhanced green fluorescent protein (EGFP), it localizes to the membrane and S. pombe carrying gt1 but not the wild-type strain can grow on medium containing glycerol as the sole carbon source. The present study is the first to report that AOX1 in the X-33gt1 mutant can achieve constitutive expression in medium containing glycerol; thus, knocking down gt1 can eliminate the glycerol repression of PAOX1 in P. pastoris. These results suggest that the glycerol transporter may participate in the process of PAOX1 inhibition in glycerol medium.