Strategies to improve CHO cell culture performance: Targeted deletion of amino acid catabolism and apoptosis genes paired with growth inhibitor supplementation.

Chinese hamster ovary (CHO) cells are the predominant host of choice for recombinant monoclonal antibody (mAb) expression. Recent advancements in gene editing technology have enabled engineering new CHO hosts with higher growth, viability, or productivity. One approach involved knock out (KO) of BCAT1 gene, which codes for the first enzyme in the branched chain amino acid (BCAA) catabolism pathway; BCAT1 KO reduced accumulation of growth inhibitory short chain fatty acid (SCFA) byproducts and improved culture growth and titer when used in conjunction with high-end pH-controlled delivery of glucose (HiPDOG) technology and SCFA supplementation during production. Accumulation of SCFAs in the culture media is critical for metabolic shift toward higher specific productivity and hence titer. Here we describe knocking out BCKDHa/b genes (2XKO), which act downstream of the BCAT1, in a BAX/BAK KO CHO host cell line background to reduce accumulation of growth-inhibitory molecules in culture. Evaluation of the new 4XKO CHO cell lines in fed-batch production cultures (without HiPDOG) revealed that partial KO of BCKDHa/b genes in an apoptosis-resistant (BAX/BAK KO) background can achieve higher viabilities and mAb titers. This was evident when SCFAs were added to boost productivity as such additives negatively impacted culture viability in the WT but not BAX/BAK KO cells during batch production. Altogether, our findings suggest that SCFA addbacks can significantly increase productivity and mAb titers in the context of apoptosis-attenuated CHO cells with partial KO of BCAA genes. Such engineered CHO hosts can offer productivity advantages for expressing biotherapeutics in an industrial setting.

Amino-terminal proteolytic fragment of the axon growth inhibitor Nogo-A (Rtn4A) is upregulated by injury and promotes axon regeneration.

After adult mammalian central nervous system injury, axon regeneration is extremely limited or absent, resulting in persistent neurological deficits. Axon regeneration failure is due in part to the presence of inhibitory proteins, including NogoA (Rtn4A), from which two inhibitory domains have been defined. When these inhibitory domains are deleted, but an amino-terminal domain is still expressed in a gene trap line, mice show axon regeneration and enhanced recovery from injury. In contrast, when there is no amino-terminal Nogo-A fragment in the setting of inhibitory domain deletion, then axon regeneration and recovery are indistinguishable from WT. These data indicated that an amino-terminal Nogo-A fragment derived from the gene trap might promote axon regeneration, but this had not been tested directly and production of this fragment without gene targeting was unclear. Here, we describe posttranslation production of an amino-terminal fragment of Nogo-A from the intact gene product. This fragment is created by proteolysis near amino acid G214-N215 and levels are enhanced by axotomy. Furthermore, this fragment promotes axon regeneration in vitro and acts cell autonomously in neurons, in contrast to the inhibitory extracellular action of other Nogo-A domains.Proteins interacting with the amino-terminal Nogo-A fragment by immunoprecipitation include HSPA8 (HSC70, HSP7C). Suppression of HSPA8 expression by shRNA decreases axon regeneration from cerebral cortical neurons and overexpression increases axon regeneration. Moreover, the amino-terminal Nogo-A fragment increases HSPA8 chaperone activity. These data provide an explanation for varied results in different gene-targeted Nogo-A mice, as well as revealing an axon regeneration promoting domain of Nogo-A.

Identification of novel polymorphism in mammary-derived growth inhibitor gene of water buffalo and its expression analysis in the mammary gland.

Mammary-derived growth inhibitor (MDGI), a member of the lipophilic family of fatty acid-binding proteins, plays an important role in the development, regulation, and differentiation of the mammary gland. The aim of the study was to identify polymorphism in the MDGI gene and its expression analysis in the mammary gland at various stages of lactation, in Indian buffalo. Nucleotide sequence analysis of MDGI gene in different breeds of riverine and swamp buffaloes revealed a total of 16 polymorphic sites and one Indel. Different transcription factor binding sites were predicted for buffalo MDGI gene promoter sequence, using online tools and in-silico analysis indicating that the SNPs in this region can impact the gene expression regulation. Phylogenetic analysis exhibited the MDGI of buffalo being closer to other ruminants like cattle, yak, sheep, and goats. Further, the expression analysis revealed that buffalo MDGI being highly expressed in well-developed mammary glands of lactating buffalo as compared to involution/non-lactating and before functional development to start the milk production stage in heifers. Stage-specific variation in expression levels signifies the important functional role of the MDGI gene in mammary gland development and milk production in buffalo, an important dairy species in Southeast Asia.

Uncovering the promiscuous activity of IL-6 proteins: A multi-dimensional analysis of phylogeny, classification and residue conservation.

The IL-6 family of cytokines, known for their pleiotropic behavior, share binding to the gp130 receptor for signal transduction with the necessity to bind other receptors. Leukemia inhibitory factor receptor is triggered by the IL-6 family proteins: leukemia inhibitory factor (LIF), oncostatin-M (OSM), cardiotrophin-1 (CT-1), ciliary neurotrophic factor (CNTF), and cardiotrophin-like cytokine factor 1 (CLCF1). Besides the conserved binding sites to the receptor, not much is known in terms of the diversity and characteristics of these proteins in different organisms. Herein, we describe the sequence analysis of LIF, OSM, and CT-1 from several organisms, and m17, a LIF ortholog found in fishes, regarding its phylogenetics, intrinsic properties, and the impact of conserved residues on structural features. Sequences were identified in seven classes of vertebrates, showing high conservation values in binding site III, but protein-dependent results on binding site II. GRAVY, isoelectric point, and molecular weight parameters were relevant to differentiate classes in each protein and to enable, for the first time and with high fidelity, the prediction of both organism class and protein type just using machine learning approaches. OSM sequences from primates showed an increased BC loop when compared to the remaining mammals, which could influence binding to OSM receptor and tune signaling pathways. Overall, this study highlights the potential of sequence diversity analysis to understand IL-6 cytokine family evolution, showing the conservation of function-related motifs and evolution of class and protein-dependent characteristics. Our results could impact future medical treatment of disorders associated with imbalances in these cytokines.

Discovery of Demurilactone A: A Specific Growth Inhibitor of L-Form Bacillus subtilis.

Metabolic profiling of the extracts from a library of actinobacteria led to the identification of a novel polyketide, demurilactone A, produced by Streptomyces strain DEM21308. The structure of the compound was assigned based on a detailed investigation of 1D/2D NMR spectra and HR-MS. Whole genome DNA sequencing, followed by bioinformatics analysis and insertional mutagenesis, identified type I polyketide synthases encoded by the dml gene cluster to direct the biosynthesis of this polyene macrolide. While the number of modules is consistent with the carbon backbone of the assigned structure, some discrepancies were identified in the domain organization of five modules. Close investigation of the amino acid sequences identified several mutations in the conserved motifs of nonfunctional domains. Furthermore, the absolute configuration of hydroxy-bearing stereocenters was proposed based on analyses of the ketoreductase domains. Remarkably, although demurilactone A has little detectable activity against normal-walled bacteria, it specifically inhibits the growth of cell wall-deficient "L-form" Bacillus subtilis at a minimum inhibitory concentration value of 16 µg/mL. Time-lapse microscopy analyses revealed that demurilactone affects membrane dynamics, probably by reducing membrane fluidity. This compound could be a powerful reagent for studying long-standing questions about the involvement of L-forms in recurrent infection.

Enhanced tolerance of Cupriavidus necator NCIMB 11599 to lignocellulosic derived inhibitors by inserting NAD salvage pathway genes.

Polyhydroxybutyrate (PHB) is a bio-based, biodegradable and biocompatible plastic that has the potential to replace petroleum-based plastics. Lignocellulosic biomass is a promising feedstock for industrial fermentation to produce bioproducts such as polyhydroxybutyrate (PHB). However, the pretreatment processes of lignocellulosic biomass lead to the generation of toxic byproducts, such as furfural, 5-HMF, vanillin, and acetate, which affect microbial growth and productivity. In this study, to reduce furfural toxicity during PHB production from lignocellulosic hydrolysates, we genetically engineered Cupriavidus necator NCIMB 11599, by inserting the nicotine amide salvage pathway genes pncB and nadE to increase the NAD(P)H pool. We found that the expression of pncB was the most effective in improving tolerance to inhibitors, cell growth, PHB production and sugar consumption rate. In addition, the engineered strain harboring pncB showed higher PHB production using lignocellulosic hydrolysates than the wild-type strain. Therefore, the application of NAD salvage pathway genes improves the tolerance of Cupriavidus necator to lignocellulosic-derived inhibitors and should be used to optimize PHB production.

Discovery of a potent cytotoxic agent that promotes G2/M phase cell cycle arrest and apoptosis in a malignant human pharyngeal squamous carcinoma cell line.

Squamous cell carcinoma is the major form of malignancy that arises in head and neck cancer. The modest improvement in the 5­year survival rate underpins its complex etiology and provides the impetus for the discovery of new therapeutics. The present study describes the discovery of an indole­based small molecule (24a) that was a potent cytotoxic agent with antiproliferative and pro­apoptotic properties against a pharyngeal carcinoma cell line, Detroit 562, effectively killing the cells at a half­maximal inhibitory concentration of 0.03 µM, as demonstrated using cell proliferation studies. The antiproliferative property of 24a was demonstrated by its ability to promote G2/M blockade, as assessed by cell cycle analysis using flow cytometry and the monitoring of real­time cell cycle progression by the fluorescence ubiquitination­based cell cycle indicator. This pro­apoptotic property is supported by the promotion of TUNEL­staining and increase in the activities of caspases­3/7 and ­6, in addition to the expression of death receptors and the cleavage of poly (ADP­ribose) polymerase 1 protein as demonstrated by western blotting. Given that Detroit 562 lacks functional p53, it is suggested that 24a acts independently of the tumor suppressor.

Different training patterns at recovery stage improve cognitive function in ischemic stroke rats through regulation of the axonal growth inhibitor pathway.

Running wheel exercise training (RWE) and skilled reaching training (SRT) are physical training approaches with positive effects on cognitive function. However, few studies have compared the different effects of these exercises on long-term memory, and their mechanism remains unknown. This study investigated the effects of SRT and RWE, at the recovery stage, on the cognitive function of transient middle cerebral artery occlusion (tMCAO) rats and explored their association with NgR1/Rho-A/ROCK/LOTUS/LGI1 signaling. Adult Sprague-Dawley rats (n = 55) were divided into four groups after pretraining: SRT, RWE, tMCAO, and Sham. Rats were subjected to modified neurological severity score (mNSS) measurements and forelimb grip strength and the Morris water maze tests. Using immunofluorescence and western blotting, we evaluated axonal growth inhibitor expression in the peri-infarct cortex on days 28 and 56 after tMCAO. Results showed the mNSS reduced, whereas the grip strengths improved in RWE and SRT groups. The escape latency in the Morris water maze test was shorter, whereas the number of times of crossing the platform was higher in both the SRT and RWE groups than in the tMCAO group on day 56; furthermore, the parameters in the SRT group improved compared to those in the RWE group. Physical exercise training could improve cognitive functions by reducing the expression of the NgR1/RhoA/ROCK axon growth inhibitors and increasing the expression of the endogenous antagonists LOTUS/LGI1. Exercise training beginning at the recovery stage could improve the cognitive function in tMCAO rats through a mechanism probably associated with the axonal growth inhibitor pathway.

A glypican-1-targeted antibody-drug conjugate exhibits potent tumor growth inhibition in glypican-1-positive pancreatic cancer and esophageal squamous cell carcinoma.

An antibody-drug conjugate (ADC) is a promising therapeutic modality because selective and effective delivery of an anti-cancer drug is achieved by drug-conjugated antibody-targeting cancer antigen. Glypican 1 (GPC1) is highly expressed in malignant tumors, including pancreatic ductal adenocarcinoma (PDAC) and esophageal squamous cell carcinoma (ESCC). Herein, we describe the usefulness of GPC1-targeting ADC. Humanized anti-GPC1 antibody (clone T2) was developed and conjugated with monomethyl auristatin E (MMAE) via maleimidocaproyl-valine-citrulline-p-aminobenzyloxycarbonyl (mc-vc-PABC) linkers (humanized GPC1-ADC[MMAE]). Humanized GPC1-ADC(MMAE) inhibited the growth of GPC1-positive PDAC and ESCC cell lines via inducing cycle arrest in the G2/M phase and apoptosis in vitro. The binding activity of humanized GPC1-ADC(MMAE) with GPC1 was comparable with that of the unconjugated anti-GPC1 antibody. The humanized GPC1-ADC(MMAE) was effective in GPC1-positive BxPC-3 subcutaneously xenografted mice but not in GPC1-negative BxPC-3-GPC1-KO xenografted mice. To assess the bystander killing activity of the humanized GPC1-ADC(MMAE), a mixture of GPC1-positive BxPC-3 and GPC1-negative BxPC-3-GPC1-KO-Luc cells were subcutaneously inoculated, and a heterogenous GPC1-expressing tumor model was developed. The humanized GPC1-ADC(MMAE) inhibited the tumor growth and decreased the luciferase signal, measured with an in vivo imaging system (IVIS), which suggests that the suppression of the BxPC-3-GPC1-KO-Luc population. The humanized GPC1-ADC(MMAE) also inhibited the established liver metastases of BxPC-3 cells and significantly improved the overall survival of the mice. It exhibited a potent antitumor effect on the GPC1-positive PDAC and ESCC patient-derived xenograft (PDX) models. Our preclinical data demonstrate that GPC1 is a promising therapeutic target for ADC.

STK3 Suppresses Ovarian Cancer Progression by Activating NF-κB Signaling to Recruit CD8+ T-Cells.

Serine/threonine protein kinase-3 (STK3) is a critical molecule of the Hippo pathway but little is known about its biological functions in the ovarian cancer development. We demonstrated the roles of STK3 in ovarian cancer. Existing databases were used to study the expression profile of STK3. STK3 was significantly downregulated in OC patients, and the low STK3 expression was correlated with a poor prognosis. In vitro cell proliferation, apoptosis, and migration assays, and in vivo subcutaneous xenograft tumor models were used to determine the roles of STK3. The overexpression of STK3 significantly inhibited cell proliferation, apoptosis, and migration of ovarian cancer cells in vitro and tumor growth in vivo. Bisulfite sequencing PCR analysis was performed to validate the methylation of STK3 in ovarian cancer. RNA sequencing and gene set enrichment analysis (GSEA) were used to compare the transcriptome changes in the STK3 overexpression ovarian cancer and control cells. The signaling pathway was analyzed by western blotting. STK3 promoted the migration of CD8+ T-cells by activating nuclear transcription factor κB (NF-κB) signaling. STK3 is a potential predictor of OC. It plays an important role in suppressing tumor growth of ovarian cancer and in chemotaxis of CD8+ T-cells.

Screening of new cell cycle suppressive compounds from marine-derived microorganisms in Chinese hamster ovary cells.

Monoclonal antibodies (mAbs) are active pharmaceutical ingredients in antibody drugs, produced mainly using recombinant Chinese hamster ovary (CHO) cells. The regulation of recombinant CHO cell proliferation can improve the productivity of heterologous proteins. Chemical compound approaches for cell cycle regulation have the advantages of simplicity and ease of use in industrial processes. However, CHO cells have genetic and phenotypic diversity, and the effects of such compounds might depend on cell line and culture conditions. Increasing the variety of cell cycle inhibitors is a promising strategy to overcome the dependency. Marine microorganisms are a vast and largely undeveloped source of secondary metabolites with physiological activity. In this study, we focused on secondary metabolites of marine microorganisms and evaluated their effectiveness as cell cycle inhibitory compounds. Of 720 extracts from microorganisms (400 actinomycetes and 320 filamentous fungi) collected from the Okinawan Sea, we identified nine extracts that decreased the specific growth rate and increased the specific production rate without reducing cell viability. After fractionating the extracts, the components of active fractions were estimated using time-of-flight mass spectrometry analysis. Then, four compounds, including staurosporine and undecylprodigiosin were deduced to be active compounds. These compounds have been reported to exert a cell cycle inhibitory effect on mammalian cells. These compounds might serve as additives to improve mAb production in CHO cells. This study indicates that secondary metabolites of marine microorganisms are a useful source for new cell cycle inhibitory compounds that can increase mAb production in CHO cells.

Iso- but Not Anteiso-Branched Chain Fatty Acids Exert Growth-Inhibiting and Apoptosis-Inducing Effects in MCF-7 Cells.

The present study compared the growth-inhibitory effects of four common branched chain fatty acids (BCFAs) found in beef and dairy fats including iso 15:0, anteiso 15:0, iso 17:0, and anteiso 17:0. MCF-7 human breast cancer cells were exposed for 72 h to media containing increasing doses (50 to -400 µM) of the four BCFA. Cell viability was not affected by any of the BCFA treatments at doses less than 200 µM. Culturing cells with 200 µM of iso-15:0 or iso-17:0 reduced cell viability by 27 ± 2.8 and 43 ± 8.3% at 24 h, 35 ± 4.6 and 49 ± 9.1% at 48 h, and 44 ± 6.8 and 57 ± 8.8% at 72 h posttreatment. In contrast, culturing cells with 200 µM of anteiso-15:0 or anteiso-17:0 did not affect cell viability for any durations tested. The incorporation of iso 15:0 and iso 17:0 into cells (19.1 ± 1.3 and 21.2 ± 1.4 µmol/mg protein, respectively) was greater (P < 0.01) than that of anteiso 15:0 and anteiso 17:0 (11.8 ± 0.7 and 13.8 ± 0.8 µmol/mg protein, respectively). Iso-15:0 and iso-17:0 downregulated (P < 0.01) the expression of antiapoptotic Bcl-2 (0.71 ± 0.6-fold and 0.64 ± 0.09-fold, respectively) and upregulated (P < 0.01) the expression of proapoptotic Bax (1.72 ± 0.14-fold and 2.15 ± 0.24-fold, respectively) compared to the control, whereas their corresponding anteiso isomers did not affect the expression of any apoptosis-related genes. Our findings suggest that the branching structure influences anticarcinogenic effects of BCFAs, with iso being more potent than anteiso.

Interaction of 4-ethylphenol, pH, sucrose and ethanol on the growth and fermentation capacity of the industrial strain of Saccharomyces cerevisiae PE-2.

Volatile phenols such as 4-ethylphenol are produced from hydroxycinnamic acids by Dekkera bruxellensis, an important yeast contaminating alcoholic fermentations. 4-ethylphenol results from the decarboxylation and reduction of p-coumaric acid, a compound found in sugarcane musts. In wine, volatile phenols are responsible by sensorial alterations whereas in the context of bioethanol fermentation, little is known about their effects on the main yeast, Saccharomyces cerevisiae. Here we evaluated the interaction of 4-ethylphenol and pH, sucrose and ethanol on the growth and fermentation capacity of the industrial strain of S. cerevisiae PE-2. A central compound rotational design was utilized to evaluate the effect of 4-ethylphenol, pH, ethanol and sucrose concentration on the yeast maximum specific growth rate (µmax) in microplate experiments in YPS medium (Yeast extract-Peptone-Sucrose), at 30 °C. Following, single-cycle fermentations in YPS medium, pH 4.5, 17% sucrose, at 30 °C, with 4-ethylphenol in concentrations of 10 and 20 mg L-1 being added at the start or after 4 h of fermentation, were carried out. 4-ethylphenol affected µmax of S. cerevisiae in situations that resemble the conditions of industrial bioethanol production, especially the low pH of the fermentation medium and the high ethanol concentration because of the anaerobic sucrose uptake. The addition of 4-ethylphenol on fermentation resulted in significant effect on the cell yeast concentration, pH and alcohol production, with significant decrease from 86% to the range of 65-74% in the fermentative efficiency. The industrial yeast S. cerevisiae PE-2 growth and fermentative capacity were affected by the presence of 4-ethylphenol, a metabolite produced by D. bruxellensis, which may contribute to explain the impact of this yeast on bioethanol industrial production.

Production, structural characterization, and antiproliferative activity of exopolysaccharide produced by Scleroderma areolatum Ehrenb with different carbon source.

The effects of different three carbon sources, that is, glucose, fructose, and sucrose, on production, molecular properties and antiproliferative activity of exopolysaccharide (EPS), were evaluated in the submerged culture of Scleroderma areolatum Ehrenb. Among carbon sources examined, the addition of sucrose maximizes the mycelia production, while fructose could maximize the EPS yield. Although the predominant carbohydrate compositions identified were gluconic acid and mannose, the monosaccharide composition of EPSs was also different significantly. FT-IR spectral analysis revealed there was no significant difference among the prominent characteristic groups in three EPSs. The molecular weight of EPSs was also affected by carbon source, being generally lower compared with that with glucose. However, all EPSs molecule existed as nearly globular shape form in aqueous solution. The variation of carbon sources also affected antiproliferative activity examined in vitro using cell proliferation assay. Fructose was optimal carbon source giving higher antiproliferative activity probably due to the relatively high contents of xylose in the EPS with low molecular weight.

Antiproliferative Activity and Cell Metabolism of Hydroxycinnamic Acids in Human Colon Adenocarcinoma Cell Lines.

In this study, we investigated the antiproliferative activity and the stability and metabolic fate of the main dietary hydroxycinnamates, using two colonic adenocarcinoma cell models (Caco-2 and SW480). Dihydrocaffeic and dihydroferulic acids were the most effective against cell proliferation in both cell lines with IC50 values of 71.7 ± 1.1 and 83.1 ± 1.1 µmol/L, respectively ( P < 0.05) in Caco-2. At 200 µmol/L, caffeic and ferulic acids inhibited SW480 proliferation by 40.8 ± 1.6 and 59.9 ± 1.3%, respectively. Hydroxycinnamic acids with a catechol-type structure were degraded in Caco-2 cell medium, resulting in the production of H2O2. Intracellular Caco-2 UDP-glucuronosyltransferases and catechol- O-methyltransferases were able to form glucuronide and methyl conjugates. However, only the sulfate conjugates were detected after incubation with SW480. In addition, simple hydroxycinnamates were released from quinic and aspartic conjugates. The remarkable effect of dihydrocaffeic and dihydroferulic acids against cell proliferation is of paramount importance, since these compounds are the main metabolites detectable at the colonic level.

Improved ethanol productivity and ethanol tolerance through genome shuffling of Saccharomyces cerevisiae and Pichia stipitis.

Genome shuffling by recursive protoplast fusion between Saccharomyces cerevisiae and Pichia stipitis also known as Scheffersomyces stipitis resulted in a promising yeast hybrid strain with superior qualities than those of the parental strains in enhancing biofuel production. Our study focused on the substrate utilization, ethanol fermentation, and ethanol tolerance of the hybrids and the parental strains. The parental strain S. cerevisiae is limited to utilize only hexose sugars, and this leads to decrease in the ethanol yield when they are subjected to ethanol production from lignocellulosic biomass which is rich in pentose sugars. To overcome this limitation, we constructed a hybrid yeast strain through genome shuffling which can assimilate all the sugars present in the fermentation medium. After two rounds of recursive protoplast fusion, there was a higher increase in substrate utilization by hybrid SP2-18 compared to parental strain S. cerevisiae. SP2-18 was able to consume 34% of xylose sugar present in the fermentation medium, whereas S. cerevisiae was not able to utilize xylose. Further, the hybrid strain SP2-18 was able to reach an ethanol productivity of 1.03 g L-1 h-1, ethanol yield 0.447 g/g, and ethanol concentration 74.65 g L-1 which was relatively higher than that of the parental strain S. cerevisiae. Furthermore, the hybrid SP2-18 was found to be stable in the production of ethanol. The random amplified polymorphic DNA profile of the yeast hybrid SP2-18 shows the polymorphism between the parental strains indicating the migration of specific sugar metabolizing genes from P. stipitis, while the maximum similarity was with the parent S. cerevisiae.

Oncostatin M-induced upregulation of SDF-1 improves Bone marrow stromal cell migration in a rat middle cerebral artery occlusion stroke model.

Bone marrow-derived mesenchymal stem cells (BMSCs) exhibit potential regenerative effects on the injured brain. However, these effects are constrained by their limited ability to migrate to the injured site. Oncostatin M (OSM) has been shown to affect the proliferation and migration of mesenchymal stem cells. Therefore, in the present study, we explored whether OSM improves BMSC migration and secretion of growth factors and cytokines in a rat middle cerebral artery occlusion (MCAO) stroke model. The effect of OSM on the proliferation and apoptosis of rat BMSCs was first assessed in vitro, and the gene and secretion levels of factors related to cell nutrition and migration, such as SDF-1 and VEGF, were detected. To further explore underlying pathways triggered by OSM, BMSCs were treated with OSM in the presence or absence of inhibitors of the STAT3 and ERK pathways. Effects of OSM on SDF-1 expression in astrocytes and BMSC migration were also evaluated. In the rat MCAO model, OSM secretion levels were detected in the brain for up to 72 h after model establishment. Ventricle injection of OSM alone or OSM combined with caudal vein graft of BMSCs was then performed in MCAO stroke rats. After 72 h, production of SDF-1 and grafted BMSCs was detected in the lesion areas of the brain, and the nerve function score was evaluated. We found that the production of OSM continually increased in the brains of MCAO rats from 12 h to 72 h. OSM significantly upregulated SDF-1 in BMSCs via the STAT3 and ERK pathways and significantly promoted the expression of VEGF and MMP-2. OSM also promoted the secretion of SDF-1 in astrocytes through the STAT3 and ERK pathways to in turn enhance BMSC migration. Combination treatment with OSM and BMSCs in MCAO rats increased the migration efficiency of BMSCs in the brain, which significantly improved neurofunctional recovery while reducing the expression of inflammatory mediators and promoting the secretion of nutrition factors. Overall, these results show that OSM is highly expressed in the brains of MCAO stroke rats and can upregulate SDF-1 to promote BMSC migration. Thus, combination treatment with OSM and BMSCs improves the graft efficiency of BMSCs and neurofunctional recovery.

Ovarian carcinoma biological nanotherapy: Comparison of the advantages and drawbacks of lipid, polymeric, and hybrid nanoparticles for cisplatin delivery.

Ovarian carcinoma is one of the most common cancers among women. The most common type of ovarian cancer is epithelial ovarian cancer and cisplatin (DDP) is one of the most interesting chemotherapeutic drugs in clinical regimens for ovarian cancer. Nanoparticles (NPs) including lipid NPs, polymeric NPs, liposomes, dendrimers, oligomers, and nanotubes were usually used for anti-cancer drug delivery. In this study, DDP loaded nanostructured lipid carriers (DDP-NLC), polymeric NPs (DDP-PNP), and lipid-polymer hybrid nanoparticles (DDP-LPN) were prepared for the evaluation in vitro and in vivo. The efficiency of these three kinds of the NPs was compared in terms of in vitro drug release, cellular uptake, in vitro cell growth inhibition, in vivo pharmacokinetics, biodistribution and in vivo antitumor in mice. The size of DDP-PNP (119.8 nm) was smaller than DDP-NLC (132.4 nm) and DDP-LPN (141.2 nm). The release of DDP from DDP-NLC was faster than DDP-PNP. Cellular uptake efficiency of DDP-NLC and DDP-LPN was significantly higher than DDP-PNP. In vivo pharmacokinetics evaluation showed that plasma concentration - time curves (AUCs) of DDP-NLC, DDP-PNP, DDP-LPN and free DDP are 128, 210, 247, and 16 mg/L h, with T1/2 of 4.4, 5.1, 5.5, and 1.7 mg/L h. DDP-LPN exhibits the highest AUC and the longest T1/2. In vivo antitumor efficacy results investigated on ovarian cancer bearing BALB/c mice model demonstrated that DDP-LPN showed the strongest antitumor effect. In vitro and in vivo studies demonstrated that DDP-NLC, DDP-PNP and DDP-LPN have different advantages due to the various evaluations. The in vivo anti-tumor results indicate that DDP-LPN may have the best tumor inhibition ability. DDP-NLC, DDP-PNP, and DDP-LPN developed in this study could be used as promising strategies for the treatment of ovarian cancer according to different demands.

IL-33 Released in the Liver Inhibits Tumor Growth via Promotion of CD4+ and CD8+ T Cell Responses in Hepatocellular Carcinoma.

IL-33 released by epithelial cells and immune cells functions as an alarmin and can induce both type 1 and type 2 immune responses. However, the role of IL-33 release in tumor development is still not clear. In this study, we examined the function of released IL-33 in murine hepatocellular carcinoma (HCC) models by hydrodynamically injecting either IL-33-expressing tumor cells or IL-33-expressing plasmids into the liver of tumor-bearing mice. Tumor growth was greatly inhibited by IL-33 release. This antitumor effect of IL-33 was dependent on suppression of tumorigenicity 2 (ST2) because it was diminished in ST2-/- mice. Moreover, HCC patients with high IL-33 expression have prolonged overall survival compared with the patients with low IL-33 expression. Further study showed that there were increased percentages and numbers of activated and effector CD4+ and CD8+ T cells in both spleen and liver in IL-33-expressing tumor-bearing mice. Moreover, IFN-γ production of the CD4+ and CD8+ T cells was upregulated in both spleen and liver by IL-33. The cytotoxicity of CTLs from IL-33-expressing mice was also enhanced. In vitro rIL-33 treatment could preferentially expand CD8+ T cells and promote CD4+ and CD8+ T cell activation and IFN-γ production. Depletion of CD4+ and CD8+ T cells diminished the antitumor activity of IL-33, suggesting that the antitumor function of released IL-33 was mediated by both CD4+ and CD8+ T cells. Taken together, we demonstrated in murine HCC models that IL-33 release could inhibit tumor development through its interaction with ST2 to promote antitumor CD4+ and CD8+ T cell responses.

Elucidating cellular mechanisms of Saccharomyces cerevisiae tolerant to combined lignocellulosic-derived inhibitors using high-throughput phenotyping and multiomics analyses.

A robust cell factory that can tolerate combined inhibitory lignocellulosic compounds is essential for the cost-effective lignocellulose-based production of second-generation bioethanol and other bulk chemicals. Following high-throughput phenotyping of a yeast genomic overexpression library, we identified a Saccharomyces cerevisiae mutant (denoted AFb.01) with improved growth and fermentation performance under combined toxicity of acetic acid and furfural. AFb.01 carries overexpression of TRX1, which encodes for thioredoxin, a cellular redox machinery. Through comparative proteomics and metabolomics, the resulting cell-wide changes in the mutant were elucidated and these primarily target on the maintenance of energy and redox homeostasis and the minimization of stress-induced cell damages. In particular, the upregulation of the stress-response proteins Hsp26p and Fmp16p conferred tolerance of AFb.01 against protein denaturation and DNA damage. Moreover, increased levels of protectant metabolites such as trehalose, fatty acids, GABA and putrescine provided additional defense mechanisms for the mutant against oxidative and redox stresses. Future studies will concentrate on targeted genetic engineering to validate these mechanisms as well as to support the creation of more robust yeast strains, applicable for industrial, cost-competitive biorefinery production.