Temporal dynamics of stress response in Halomonas elongata to NaCl shock: physiological, metabolomic, and transcriptomic insights.

BACKGROUND: The halophilic bacterium Halomonas elongata is an industrially important strain for ectoine production, with high value and intense research focus. While existing studies primarily delve into the adaptive mechanisms of this bacterium under fixed salt concentrations, there is a notable dearth of attention regarding its response to fluctuating saline environments. Consequently, the stress response of H. elongata to salt shock remains inadequately understood. RESULTS: This study investigated the stress response mechanism of H. elongata when exposed to NaCl shock at short- and long-time scales. Results showed that NaCl shock induced two major stresses, namely osmotic stress and oxidative stress. In response to the former, within the cell's tolerable range (1-8% NaCl shock), H. elongata urgently balanced the surging osmotic pressure by uptaking sodium and potassium ions and augmenting intracellular amino acid pools, particularly glutamate and glutamine. However, ectoine content started to increase until 20 min post-shock, rapidly becoming the dominant osmoprotectant, and reaching the maximum productivity (1450 ± 99 mg/L/h). Transcriptomic data also confirmed the delayed response in ectoine biosynthesis, and we speculate that this might be attributed to an intracellular energy crisis caused by NaCl shock. In response to oxidative stress, transcription factor cysB was significantly upregulated, positively regulating the sulfur metabolism and cysteine biosynthesis. Furthermore, the upregulation of the crucial peroxidase gene (HELO_RS18165) and the simultaneous enhancement of peroxidase (POD) and catalase (CAT) activities collectively constitute the antioxidant defense in H. elongata following shock. When exceeding the tolerance threshold of H. elongata (1-13% NaCl shock), the sustained compromised energy status, resulting from the pronounced inhibition of the respiratory chain and ATP synthase, may be a crucial factor leading to the stagnation of both cell growth and ectoine biosynthesis. CONCLUSIONS: This study conducted a comprehensive analysis of H. elongata's stress response to NaCl shock at multiple scales. It extends the understanding of stress response of halophilic bacteria to NaCl shock and provides promising theoretical insights to guide future improvements in optimizing industrial ectoine production.

Uncovering the Role of Hydroxycinnamoyl Transferase in Boosting Chlorogenic Acid Accumulation in Carthamus tinctorius Cells under Methyl Jasmonate Elicitation.

Chlorogenic acids (CGAs) are bioactive compounds widely used in the food, pharmaceutical, and cosmetic industries. Carthamus tinctorius is an important economic crop, and its suspension cells are rich in CGAs. However, little is known about the biosynthesis and regulation of CGAs in Carthamus tinctorius cells. This study first elucidated the regulatory mechanism of CGA biosynthesis in methyl jasmonate (MeJA)-treated Carthamus tinctorius cells and the role of the MeJA-responsive hydroxycinnamoyl transferase (HCT) gene in enhancing their CGA accumulation. Firstly, temporal changes in intracellular metabolites showed that MeJA increased the intracellular CGA content up to 1.61-fold to 100.23 mg·g-1. Meanwhile, 31 primary metabolites showed significant differences, with 6 precursors related to increasing CGA biosynthesis. Secondly, the transcriptome data revealed 3637 new genes previously unannotated in the Carthamus tinctorius genome and 3653 differentially expressed genes. The genes involved in the plant signaling pathway and the biosynthesis of CGAs and their precursors showed a general up-regulation, especially the HCT gene family, which ultimately promoted CGA biosynthesis. Thirdly, the expression of a newly annotated and MeJA-responsive HCT gene (CtHCT, CtNewGene_3476) was demonstrated to be positively correlated with CGA accumulation in the cells, and transient overexpression of CtHCT enhanced CGA accumulation in tobacco. Finally, in vitro catalysis kinetics and molecular docking simulations revealed the ability and mechanism of the CtHCT protein to bind to various substrates and catalyze the formation of four hydroxycinnamic esters, including CGAs. These findings strengthened our understanding of the regulatory mechanism of CGA biosynthesis, thereby providing theoretical support for the efficient production of CGAs.

[Effects of manipulating lactate dehydrogenase gene on metabolism of HEK-293 and production of human adenovirus].

Reducing lactate accumulation has always been a goal of the mammalian cell biotechnology industry. When animal cells are cultured in vitro, the accumulation of lactate is mainly the combined result of two metabolic pathways. On one hand, glucose generates lactate under the function of lactate dehydrogenase A (LDHA); on the other hand, lactate can be oxidized to pyruvate by LDHB or LDHC and re-enter the TCA cycle. This study comprehensively evaluated the effects of LDH manipulation on the growth, metabolism and human adenovirus (HAdV) production of human embryonic kidney 293 (HEK-293) cells, providing a theoretical basis for engineering the lactate metabolism in mammalian cells. By knocking out ldha gene and overexpression of ldhb and ldhc genes, the metabolic efficiency of HEK-293 cells was effectively improved, and HAdV production was significantly increased. Compared with the control cell, LDH manipulation promoted cell growth, reduced the accumulation of lactate and ammonia, significantly enhanced the efficiency of substrate and energy metabolism of cells, and significantly increased the HAdV production capacity of HEK-293 cells. Among these LDH manipulation measures, ldhc gene overexpression performed the best, with the maximum cell density increased by about 38.7%. The yield of lactate to glucose and ammonia to glutamine decreased by 33.8% and 63.3%, respectively; and HAdV titer increased by at least 16 times. In addition, the ATP production rate, ATP/O2 ratio, ATP/ADP ratio and NADH content of the modified cell lines were increased to varying degrees, and the energy metabolic efficiency was significantly improved.

A systematic evaluation of quenching and extraction procedures for quantitative metabolome profiling of HeLa carcinoma cell under 2D and 3D cell culture conditions.

Metabolic reprogramming has been coined as a hallmark of cancer, accompanied by which the alterations in metabolite levels have profound effects on gene expression, cellular differentiation, and the tumor environment. Yet a systematic evaluation of quenching and extraction procedures for quantitative metabolome profiling of tumor cells is currently lacking. To achieve this, this study is aimed at establishing an unbiased and leakage-free metabolome preparation protocol for HeLa carcinoma cell. We evaluated 12 combinations of quenching and extraction methods from three quenchers (liquid nitrogen, -40°C 50% methanol, 0.5°C normal saline) and four extractants (-80°C 80% methanol, 0.5°C methanol/chloroform/water [1:1:1 v/v/v], 0.5°C 50% acetonitrile, 75°C 70% ethanol) for global metabolite profiling of adherent HeLa carcinoma cells. Based on the isotope dilution mass spectrometry (IDMS) method, gas/liquid chromatography in tandem with mass spectrometry was used to quantitatively determine 43 metabolites including sugar phosphates, organic acids, amino acids (AAs), adenosine nucleotides, and coenzymes involved in central carbon metabolism. The results showed that the total amount of the intracellular metabolites in cell extracts obtained using different sample preparation procedures with the IDMS method ranged from 21.51 to 295.33 nmol per million cells. Among 12 combinations, cells that washed twice with phosphate buffered saline (PBS), quenched with liquid nitrogen, and then extracted with 50% acetonitrile were found to be the most optimal method to acquire intracellular metabolites with high efficiency of metabolic arrest and minimal loss during sample preparation. In addition, the same conclusion was drawn as these 12 combinations were applied to obtain quantitative metabolome data from three-dimensional (3D) tumor spheroids. Furthermore, a case study was carried out to evaluate the effect of doxorubicin (DOX) on both adherent cells and 3D tumor spheroids using quantitative metabolite profiling. Pathway enrichment analysis using targeted metabolomics data showed that DOX exposure would significantly affect AA metabolism-related pathways, which might be related to the mitigation of redox stress. Strikingly, our data suggested that compared to two-dimensional (2D) cells the increased intracellular glutamine level in 3D cells benefited replenishing the tricarboxylic acid (TCA) cycle when the glycolysis was limited after dosing with DOX. Taken together, this study provides a well-established quenching and extraction protocol for quantitative metabolome profiling of HeLa carcinoma cell under 2D and 3D cell culture conditions. Based on this, quantitative time-resolved metabolite data can serve to the generation of hypotheses on metabolic reprogramming to reveal its important role in tumor development and treatment.

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.

Engineered Bacteria-Based Living Materials for Biotherapeutic Applications.

Future advances in therapeutics demand the development of dynamic and intelligent living materials. The past static monofunctional materials shall be unable to meet the requirements of future medical development. Also, the demand for precision medicine has increased with the progressively developing human society. Therefore, engineered living materials (ELMs) are vitally important for biotherapeutic applications. These ELMs can be cells, microbes, biofilms, and spores, representing a new platform for treating intractable diseases. Synthetic biology plays a crucial role in the engineering of these living entities. Hence, in this review, the role of synthetic biology in designing and creating genetically engineered novel living materials, particularly bacteria, has been briefly summarized for diagnostic and targeted delivery. The main focus is to provide knowledge about the recent advances in engineered bacterial-based therapies, especially in the treatment of cancer, inflammatory bowel diseases, and infection. Microorganisms, particularly probiotics, have been engineered for synthetic living therapies. Furthermore, these programmable bacteria are designed to sense input signals and respond to disease-changing environments with multipronged therapeutic outputs. These ELMs will open a new path for the synthesis of regenerative medicines as they release therapeutics that provide in situ drug delivery with lower systemic effects. In last, the challenges being faced in this field and the future directions requiring breakthroughs have been discussed. Conclusively, the intent is to present the recent advances in research and biomedical applications of engineered bacteria-based therapies during the last 5 years, as a novel treatment for uncontrollable diseases.

Urchin-like magnetic microspheres for cancer therapy through synergistic effect of mechanical force, photothermal and photodynamic effects.

BACKGROUND: Magnetic materials mediated by mechanical forces to combat cancer cells are currently attracting attention. Firstly, the magnetic force penetrates deeper into tissues than the NIR laser alone to destroy tumours. Secondly, the synergistic effect of nano-magnetic-material characteristics results in a viable option for the targeted killing of cancer cells. Therefore, mechanical force (MF) produced by magnetic nanomaterials under low frequency dynamic magnetic field combined with laser technology is the most effective, safe and efficient tool for killing cancer cells and tumour growth. RESULTS: In this study, we synthesized novel urchin-like hollow magnetic microspheres (UHMMs) composed of superparamagnetic Fe3O4. We demonstrated the excellent performance of UHMMs for killing laryngocarcinoma cancer cells through mechanical force and photothermal effects under a vibrating magnetic field and near-infrared laser, respectively. The killing efficiency was further improved after loading the synthesised UHMMs with Chlorin e6 relative to unloaded UHMMs. Additionally, in animal experiments, laryngocarcinoma solid tumour growth was effectively inhibited by UHMMs@Ce6 through magneto-mechanic force, photothermal and photodynamic therapy. CONCLUSIONS: The biocompatibility and high efficiency of multimodal integrated therapy with the UHMMs prepared in this work provide new insights for developing novel nano therapy and drug loading platforms for tumour treatment. In vivo experiments further demonstrated that UHMMs/Ce6 are excellent tools for strongly inhibiting tumour growth through the above-mentioned characteristic effects.

Treatment of experimental autoimmune encephalomyelitis using AAV gene therapy by blocking T cell costimulatory pathways.

Multiple sclerosis (MS) is a chronic autoimmune disease of the central nervous system (CNS), characterized by inflammation and demyelination. Presently, repeated relapses of MS necessitate long-term immune-regulatory therapy. Blocking the CD28-B7 and CD40-CD40L costimulatory pathways is an effective and synergistic method for the prevention and amelioration of clinical symptoms of experimental autoimmune encephalomyelitis (EAE), a mouse model of MS. In this study, to explore the efficacy and safety of MS gene therapy, we used adeno-associated virus (AAV) as a vector to deliver CTLA4-immunoglobulin (Ig) or CD40-Ig on the EAE induced by myelin oligodendrocyte glycoprotein (MOG). Our results showed that a single administration of AAV8-CTLA4-Ig, either alone or with AAV8-CD40-Ig, protected mice from EAE and reversed disease progression. Decreased CD4+ and CD8+ T cell infiltration, inhibition of MOG antibody response, and downregulation of neuroinflammation were observed in mice receiving AAV, suggesting that autoimmunity was suppressed in EAE pathology. Moreover, no hematological or hepatic toxicity was observed in AAV-treated mice. Thus, compared with treatment with recombinant CTLA4-Ig (belatacept), AAV gene therapy could effectively control clinical symptoms and suppress autoimmunity in the long term. In summary, our study provides a potential therapeutic method for blocking T cell costimulation for the treatment of MS via gene therapy.

Repeated Systemic Dosing of Adeno-Associated Virus Vectors in Immunocompetent Mice After Blockade of T Cell Costimulatory Pathways.

Neutralizing antibodies (NAbs) strongly limit adeno-associated virus (AAV) vector transduction and repeated administration. Previous studies have shown that NAbs induced by AAVs are associated with T and B cell activation and that the B7/CD28 and CD40/CD40L costimulation signaling pathways are involved. Cytotoxic T lymphocyte-associated antigen 4 (CTLA4) and CD40 are vital molecules that participate in the costimulatory pathway. In this study, we evaluated CTLA4-Ig and CD40-Ig immunosuppreve efficacies through AAV and investigated their effects on the feasibility for multiple systemic administrations of AAV vectors. The results showed that a single administration of AAV vector carrying either CTLA4-Ig alone or with CD40-Ig could greatly reduce the level of NAbs. An AAV serotype-specific immune tolerance could be successfully established, which enabled repeated, that is, second and third, systemic administration of AAV vectors in the same mice. A combination of CTLA4-Ig and CD40-Ig delivered via AAV vectors significantly inhibited T and B cell activations without affecting the immune response to the total immunoglobulin G production and cytokines. Interestingly, exogenous gene expression significantly improved after multiple administrations of AAV vector in vivo. Our study generates a reliable and effective method for repeated dosing of AAV vectors that is needed on gene therapy.

Metabolic Engineering of Gas-Fermenting Clostridium ljungdahlii for Efficient Co-production of Isopropanol, 3-Hydroxybutyrate, and Ethanol.

Rational design and modification of autotrophic bacteria to efficiently produce high-value chemicals and biofuels are crucial for establishing a sustainable and economically viable process for one-carbon (C1) source utilization, which, however, remains a challenge in metabolic engineering. In this study, autotrophic Clostridium ljungdahlii was metabolically engineered to efficiently co-produce three important bulk chemicals, isopropanol, 3-hydroxybutyrate (3-HB), and ethanol (together, IHE), using syngas (CO2/CO). An artificial isopropanol-producing pathway was first constructed and optimized in C. ljungdahlii to achieve an efficient production of isopropanol and an unexpected product, 3-HB. Based on this finding, an endogenous active dehydrogenase capable of converting acetoacetate to 3-HB was identified in C. ljungdahlii, thereby revealing an efficient 3-HB-producing pathway. The engineered strain was further optimized to reassimilate acetic acid and synthesize 3-HB by introducing heterologous functional genes. Finally, the best-performing strain was able to produce 13.4, 3.0, and 28.4 g/L of isopropanol, 3-HB, and ethanol, respectively, in continuous gas fermentation. Therefore, this work represents remarkable progress in microbial production of bulk chemicals using C1 gases.

Statistical Optimizing of Medium for Clavulanic Acid Production by Streptomyces clavuligerus Using Response Surface Methodology.

Clavulanic acid (CA) is a naturally occurring antibiotic produced by Streptomyces clavuligerus. Statistical optimization of the fermentation medium for CA production by Streptomyces clavuligerus was carried out. Multiple carbon sources, glycerol, dextrin, and triolein, were considered simultaneously. A two-level fractional factorial design experiment was conducted to identify the significant components of medium on CA production. Statistical analysis of the results showed that soybean meal, dextrin, and triolein were the most significant medium ingredients on CA production. The optimal level of these screened components was obtained by RSM based on the result of a Box-Behnken design, in which the values of dextrin, soybean meal, and triolein in CA fermentation medium were 12.37 g/L, 39.75 g/L, and 26.98 ml/L, respectively. Using the proposed optimized medium, the model predicted 938 mg/L of CA level and via experimental rechecking the model, 946 mg/L of CA level was attained in shake flask fermentation, significantly high than 630 mg/L of original medium. The optimized medium was further verified in 50-L stirred fermenter, and compared with performance of original medium in parallel, CA titer was increased from 889 to 1310 mg/L; a 47% increase was achieved through medium optimization by statistical approaches.

Sandwich-Type Near-Infrared Conjugated Polymer Nanoparticles for Revealing the Fate of Transplanted Human Umbilical Cord Mesenchymal Stem Cells.

Near-infrared conjugated polymer nanoparticles (NIR-CPNs) have been widely used in in vivo imaging fields. However, most of them face the aggregation-induced fluorescence quenching (ACQ) dilemma and serious dye leakage behavior, which impedes the long-term monitoring of transplanted cells in vivo. In the present work, a novel strategy of sandwich-type encapsulation of the conjugated polymer interlayer in the crystalline SiO2 core + shell (SSiO2@SPFTBT@CSiO2) is developed, which works well to avoid the ACQ problem by homogeneously dispersing poly((9,9-dioctylfluorene-2,7-diyl)-alt-(4,7-di(thiophene-2-yl)-2,1,3-benzothiadiazole)-5',5″-diyl) (PFTBT) and suppressing intermolecular π-π stacking. Furthermore, the unparalleled nanostructure efficiently stabilizes nanoparticles and successfully achieves long-term biocompatibility without interfering the biological characteristics of stem cells, indicating the potential of SSiO2@SPFTBT@CSiO2 in cell labeling. In addition, the fate of human umbilical cord mesenchymal stem cells (hucMSCs) in a mouse model with acute liver injury was disclosed. We found that the hucMSCs mainly migrated from the lungs to the injured liver and most transplanted hucMSCs were cleared up by the liver at 8 days post-injection. Revelation of the shuttle process and period will benefit in improving the clinical efficacy of hucMSCs, and the sandwich-type encapsulation strategy could also open a new avenue to obtain bright and robust NIR-CPNs for long-term fluorescence imaging.

Multi-omics analysis defines 5-fluorouracil drug resistance in 3D HeLa carcinoma cell model.

Cervical cancer is a serious health problem in women around the globe. However, the use of clinical drug is seriously dampened by the development of drug resistance. Efficient in vitro tumor model is essential to improve the efficiency of drug screening and the accuracy of clinical application. Multicellular tumor spheroids (MTSs) can in a way recapitulates tumor traits in vivo, thereby representing a powerful transitional model between 2D monolayer culture and xenograft. In this study, based on the liquid overlay method, a protocol for rapid generation of the MTSs with uniform size and high reproducibility in a high-throughput manner was established. As expected, the cytotoxicity results showed that there was enhanced 5-fluorouracil (5-FU) resistance of HeLa carcinoma cells in 3D MTSs than 2D monolayer culture with a resistance index of 5.72. In order to obtain a holistic view of the molecular mechanisms that drive 5-FU resistance in 3D HeLa carcinoma cells, a multi-omics study was applied to discover hidden biological regularities. It was observed that in the 3D MTSs mitochondrial function-related proteins and the metabolites of the tricarboxylic acid cycle (TCA cycle) were significantly decreased, and the cellular metabolism was shifted towards glycolysis. The differences in the protein synthesis, processing, and transportation between 2D monolayer cultures and 3D MTSs were significant, mainly in the heat shock protein family, with the up-regulation of protein folding function in endoplasmic reticulum (ER), which promoted the maintenance of ER homeostasis in the 3D MTSs. In addition, at the transcript and protein level, the expression of extracellular matrix (ECM) proteins (e.g., laminin and collagen) were up-regulated in the 3D MTSs, which enhanced the physical barrier of drug penetration. Summarizing, this study formulates a rapid, scalable and reproducible in vitro model of 3D MTS for drug screening purposes, and the findings establish a critical role of glycolytic metabolism, ER hemostasis and ECM proteins expression profiling in tumor chemoresistance of HeLa carcinoma cells towards 5-FU.

Mapping molecular pathways for embryonic Sertoli cells derivation based on differentiation model of mouse embryonic stem cells.

BACKGROUND: Embryonic Sertoli cells (eSCs) have been known for playing important roles in male reproductive development system. In current studies, eSCs were mainly generated from induced intermediate mesoderm. The deriving mechanism of eSCs has been unclear so far. Therefore, this work was aimed to reveal the molecular pathways during derivation of eSCs. METHODS: In this scenario, a differentiation model from mouse embryonic stem cells (mESCs) to eSCs was established through spatiotemporal control of 5 key factors, Wilms tumor 1 homolog (Wt1), GATA binding protein 4 (Gata4), nuclear receptor subfamily 5, group A, member 1 (Nr5a1, i.e., Sf1), SRY (sex determining region Y)-box 9 (Sox9), doublesex, and mab-3 related transcription factor 1 (Dmrt1). To investigate the molecular mechanism, these key factors were respectively manipulated through a light-switchable (light-on) system, tetracycline-switchable (Tet-on) system, and CRISPR/Cas9 knock out (KO) system. RESULTS: Via the established approach, some embryonic Sertoli-like cells (eSLCs) were induced from mESCs and formed ring-like or tubular-like structures. The key factors were respectively manipulated and revealed their roles in the derivation of these eSLCs. Based on these results, some molecular pathways were mapped during the development of coelomic epithelial somatic cells to eSCs. CONCLUSIONS: This differentiation model provided a high controllability of some key factors and brought a novel insight into the deriving mechanism of Sertoli cells.

Evaluation of a panel of very long-chain lysophosphatidylcholines and acylcarnitines for screening of X-linked adrenoleukodystrophy in China.

BACKGROUND: Elevated blood C24:0- and C26:0-carnitines and lysophosphatidylcholines (LPCs) were reported as diagnostic biomarkers for X-linked adrenoleukodystrophy (X-ALD). Our aim was to establish the reference intervals of very long-chain (VLC) acylcarnitines (C20-C26) and LPCs in Chinese population, and evaluate valuable biomarkers and develop panel for screening X-ALD in China. METHODS: The method of FIA-MS/MS-based quantification of VLC acylcarnitines and LPCs was validated in order to determine their concentrations in dried blood spots from 7 X-ALD boys, 396 age-matched healthy controls, and 3078 putative normal newborns. Screening performance of these metabolites for X-ALD was clinically evaluated. RESULTS: The reference intervals of VLC acylcarnitines, LPCs and their ratios were established in Chinese population, and for some metabolites like C26 and C26:0-LPC, the reference intervals were found to be significantly different between children and newborns. C24 and C26, C26:0-LPC, C24/C22 and C26/C22 ratios were found to have better performance than other analytes to identify X-ALD boys from normal children. CONCLUSION: C26:0-LPC, C24 and C26 are three most valuable biomarkers for screening of X-ALD in children group. The information of age-related variations in concentration of some biomarkers is helpful for accurate screening of X-ALD.

Coupling metabolomics analysis and DOE optimization strategy towards enhanced IBDV production by chicken embryo fibroblast DF-1 cells.

Infectious bursal disease (IBD) caused by IBD virus (IBDV) is highly contagious viral and vaccination in chicken embryo has been an effective mean to prevent acute infection. However, the current production of IBDV vaccine faces serious batch instability and external contamination. The chicken embryonic fibroblast cell line DF-1 is widely used for the proliferation of avian viruses and vaccine production. Thus, optimizing the production of IBDV by DF-1 cells has an important application value. Combining metabolomics analysis and a Design of Experiments (DOE) statistical strategy, this study successfully optimized the process of IBDV production by DF-1 cells. Differential analysis and time series analysis of metabolite data in both IBDV-infected and uninfected DF-1 cells were performed by multivariate statistical analysis. The results showed that the intracellular metabolite intensities of glycolysis, the pentose phosphate pathway, the nucleoside synthesis pathway, lipid metabolism, and glutathione metabolism were upregulated, and the TCA cycle underwent a slight downregulation after IBDV infection of DF-1 cells. Based on the metabolome results and DOE statistical optimization method, the additive components suitable for IBDV proliferation were determined. The IBDV titer increased by 20.7 times upon exogenous addition of cysteine, methionine, lysine and nucleosides in the control medium, which is consistent with the predicted result (20.0 times) by a multivariate quadratic equation. This study provides a strategy for the efficient production of IBDV vaccines and could potentially be utilized to improve the production of other viral vaccines and biologics.

A study on enhanced O-glycosylation strategy for improved production of recombinant human chorionic gonadotropin in Chinese hamster ovary cells.

Human chorionic gonadotropin (hCG) is a glycoprotein hormone that exists as a heterodimer comprised of an α subunit and ß subunit linked with disulfide bridges. The ß subunit contains four O-glycosylation sites. Previous studies have found that the translation of mRNA to polypeptides of the ß subunit was a severely limiting step for the expression of recombinant hCG protein in Chinese hamster ovary (CHO) cells. The effects of O-glycosylation on recombinant hCG protein expression were assessed by adding O-glycan precursors and overexpressing and knocking down key regulatory genes of O-glycan precursor synthesis and O-glycan sugar chain synthesis or hydrolases. The results indicated that O-glycosylation was indeed limiting in the expression of recombinant hCG protein, and N-acetylgalactosamine (GalNAc) was the major limiting precursor. Glutamine-fructose-6-phosphate transaminase 2 (Gfat2) and Uridine diphosphate-glucose pyrophosphorylase 2 (Ugp2), key regulatory genes of O-glycan precursor synthesis, were overexpressed. Ugp2 overexpression significantly increased the recombinant hCG protein level by 1.92 times compared to that of the control. The LC-MS/MS analysis and Phaseolus vulgaris leucoagglutinin (PHA-L) lectin blot analysis showed that Ugp2 overexpression significantly increased the total galactosylation levels of intracellular proteins and the O-glycosylation of recombinant hCG protein. The stability of the hCG protein to trypsin digestion was also enhanced. Ugp2 is the major limiting enzyme of the O-glycan precursor synthesis in recombinant hCG protein production. Furthermore, the effects and mechanisms of the key genes of O-glycan sugar chain synthesis and hydrolases such as polypeptide N-acetylgalactosaminyltransferase1 (Galnt1), Core 1 synthase, glycoprotein-N-acetylgalactosamine 3-beta-galactosyltransferase (C1galt1), O-linked N-acetylglucosamine transferase (Ogt) and Hexosaminidase (Hex), were evaluated. The results indicated that Galnt1 overexpression increased the recombinant hCG protein level by 1.57 times and improved the total galactosylation of intracellular proteins, O-glycosylation and the stability of recombinant hCG protein. Galnt1 is the major limiting enzyme of O-glycan sugar chain synthesis. Overexpression of Ugp2 and Galnt1 simultaneously improved the recombinant hCG protein level by 2.44 times, and both had synergistic effects. Based on the results of overexpression of Galnt1, the major limiting gene of O-Glycan chain synthesis, the precursors GalNAc and Gal were added and increased the recombinant hCG protein level by 3.68 times. This study revealed the major limiting factors of O-glycosylation of recombinant hCG protein in CHO cells and proposed an effective expression regulation strategy.

Combinatorial Effect of ARTP Mutagenesis and Ribosome Engineering on an Industrial Strain of Streptomyces albus S12 for Enhanced Biosynthesis of Salinomycin.

Salinomycin, an important polyketide, has been widely utilized in agriculture to inhibit growth of pathogenic bacteria. In addition, salinomycin has great potential in treatment of cancer cells. Due to inherited characteristics and beneficial potential, its demand is also inclining. Therefore, there is an urgent need to increase the current high demand of salinomycin. In order to obtain a high-yield mutant strain of salinomycin, the present work has developed an efficient breeding process of Streptomyces albus by using atmospheric and room temperature plasma (ARTP) combined with ribosome engineering. In this study, we investigate the presented method as it has the advantage of significantly shortening mutant screening duration by using an agar block diffusion method, as compared to other traditional strain breeding methods. As a result, the obtained mutant Tet30Chl25 with tetracycline and chloramphenicol resistance provided a salinomycin yield of 34,712 mg/L in shake flask culture, which was over 2.0-fold the parental strain S12. In addition, comparative transcriptome analysis of low and high yield mutants, and a parental strain revealed the mechanistic insight of biosynthesis pathways, in which metabolic pathways including butanoate metabolism, starch and sucrose metabolism and glyoxylate metabolism were closely associated with salinomycin biosynthesis. Moreover, we also confirmed that enhanced flux of glyoxylate metabolism via overexpression gene of isocitrate lyase (icl) promoted salinomycin biosynthesis. Based on these results, it has been successfully verified that the overexpression of crotonyl-CoA reductase gene (crr) and transcriptional regulator genes (orf 3 and orf 15), located in salinomycin synthesis gene cluster, is possibly responsible for the increase in salinomycin production in a typical strain Streptomyces albus DSM41398. Conclusively, a tentative regulatory model of ribosome engineering combined with ARTP in S. ablus is proposed to explore the roles of transcriptional regulators and stringent responses in the biosynthesis regulation of salinomycin.

Comparative Fluxome and Metabolome Analysis of Formate as an Auxiliary Substrate for Penicillin Production in Glucose-Limited Cultivation of Penicillium chrysogenum.

During glucose-limited growth, a substantial input of adenosine triphosphate (ATP) is required for the production of ß-lactams by the filamentous fungus Penicillium chrysogenum. Formate dehydrogenase has been confirmed in P. chrysogenum for formate oxidation allowing an extra supply of ATP, and coassimilation of glucose and formate has the potential to increase penicillin production and biomass yield. In this study, the steady-state metabolite levels and fluxes in response to cofeeding of formate as an auxiliary substrate in glucose-limited chemostat cultures at the dilution rates (D) of both 0.03 h-1 and 0.05 h-1 are determined to evaluate the quantitative impact on the physiology of a high-yielding P. chrysogenum strain. It is observed that an equimolar addition of formate is conducive to an increase in both biomass yield and penicillin production at D = 0.03 h-1 , while this is not the case at D = 0.05 h-1 . In addition, a higher cytosolic redox status (NADH/NAD+ ), a higher intracellular glucose level, and lower penicillin productivity are only observed upon formate addition at D = 0.05 h-1 , which are virtually absent at D = 0.03 h-1 . In conclusion, the results demonstrate that the effect of formate as an auxiliary substrate on penicillin productivity in the glucose-limited chemostat cultivations of P. chrysogenum is not only dependent on the formate/glucose ratio as published before but also on the specific growth rate. The results also imply that the overall process productivity and quality regarding the use of formate should be further explored in an actual industrial-scale scenario.

Novel Piperazino-Enaminones Decrease Pro-inflammatory Cytokines Following Hemarthrosis in a Hemophilia Mouse Model.

Hemarthrosis is the primary cause of hemophiliac arthropathy (HA). Pro-inflammatory cytokines are thought to play an important role in the pathogenesis of HA, and thus, anti-cytokine approaches may be used as an adjuvant therapy. A novel series of enaminone compounds (JODI), that contain the N-aryl piperazino motif, have been shown in vitro to reduce pro-inflammatory cytokines and thus may be efficacious in vivo. In this report, we will assess whether JODI can suppress multiple cytokines which might be potentially responsible for joint inflammation in a mouse model of hemarthrosis. The results showed that JODI significantly improved the survival after LPS treatment, and most pro-inflammatory cytokines/chemokines were decreased significantly after JODI administration. In the hemophilia mouse model, hemarthrosis resulted in local cytokine/chemokine changes, represented by elevated pro-inflammatory (IL-6, MCP-1, MIP-1α, MIP-1ß) and pro-angiogenic (VEGF and IL-33) cytokines, and decreased anti-pro-inflammatory cytokines IL-4 and IL-10. The changes were reversed by administration of JODI, which can be used as a novel approach to manage hemophilia arthropathy.