Mechanistic insights into inositol-mediated rumen function promotion and metabolic alteration using in vitro and in vivo models.

Inositol is a bioactive factor that is widely found in nature; however, there are few studies on its use in ruminant nutrition. This study investigated the effects of different inositol doses and fermentation times on rumen fermentation and microbial diversity, as well as the levels of rumen and blood metabolites in sheep. Rumen fermentation parameters, microbial diversity, and metabolites after different inositol doses were determined in vitro. According to the in vitro results, six small-tailed Han sheep fitted with permanent rumen fistulas were used in a 3 × 3 Latin square feeding experiment where inositol was injected into the rumen twice a day and rumen fluid and blood samples were collected. The in vitro results showed that inositol could increase in vitro dry matter digestibility, in vitro crude protein digestibility, NH3-N, acetic acid, propionic acid, and rumen microbial diversity and affect rumen metabolic pathways (p < 0.05). The feeding experiment results showed that inositol increased the blood concentration of high-density lipoprotein and IgG, IgM, and IL-4 levels. The rumen microbial composition was significantly affected (p < 0.05). Differential metabolites in the rumen were mainly involved in ABC transporters, biotin metabolism, and phenylalanine metabolism, whereas those in the blood were mainly involved in arginine biosynthesis and glutathione and tyrosine metabolism. In conclusion, inositol improves rumen function, affects rumen microorganisms and rumen and blood metabolites and may reduce inflammation, improving animal health.

Impact of deoxynivalenol on rumen function, production, and health of dairy cows: Insights from metabolomics and microbiota analysis.

Deoxynivalenol contamination in feed and food, pervasive from growth, storage, and processing, poses a significant risk to dairy cows, particularly when exposed to a high-starch diet; however, whether a high-starch diet exacerbates these negative effects remains unclear. Therefore, we investigated the combined impact of deoxynivalenol and dietary starch on the production performance, rumen function, and health of dairy cows using metabolomics and 16 S rRNA sequencing. Our findings suggested that both high- and low-starch diets contaminated with deoxynivalenol significantly reduced the concentration of propionate, isobutyrate, valerate, total volatile fatty acids (TVFA), and microbial crude protein (MCP) concentrations, accompanied by a noteworthy increase in NH3-N concentration in vitro and in vivo (P < 0.05). Deoxynivalenol altered the abundance of microbial communities in vivo, notably affecting Oscillospiraceae, Lachnospiraceae, Desulfovibrionaceae, and Selenomonadaceae. Additionally, it significantly downregulated lecithin, arachidonic acid, valine, leucine, isoleucine, arginine, and proline metabolism (P < 0.05). Furthermore, deoxynivalenol triggered oxidative stress, inflammation, and dysregulation in immune system linkage, ultimately compromising the overall health of dairy cows. Collectively, both high- and low-starch diets contaminated with deoxynivalenol could have detrimental effects on rumen function, posing a potential threat to production performance and the overall health of cows. Notably, the negative effects of deoxynivalenol are more pronounced with a high-starch diet than a low-starch diet.

Prognostic and immunological potential of PPM1G in lung adenocarcinoma.

Lung cancer is one of the most common types of cancer worldwide, with the highest incidence and mortality rates. Protein phosphatase, Mg2+/Mn2+ dependent 1G (PPM1G) is a serine/threonine phosphatase, which is involved in the proliferation, invasion and metastasis of tumor cells. However, there are few reports on the role of PPM1G in lung adenocarcinoma (LUAD). The present study used publicly available data from The Cancer Genome Atlas (TCGA) and Gene Expression Omnibus databases to evaluate the expression of PPM1G in LUAD, and to assess the relationship between PPM1G expression and the prognosis of patients with LUAD. Protein expression data of PPM1G obtained by immunohistochemical staining were collected from the Human Protein Atlas database. The correlation between PPM1G and immune cell infiltration and immune checkpoints was analyzed by single­sample gene set enrichment analysis of TCGA data. The Kaplan­Meier method was used for survival analysis, and univariate and multivariate Cox regression were used to analyze the effect of PPM1G on prognosis with data from TCGA database. The results showed that PPM1G was highly expressed in LUAD cancer tissues. The high expression of PPM1G was associated with poor clinical stage, T stage, N stage and overall survival in LUAD. The present study screened 29 genes related to PPM1G and closely related to the cell cycle in patients with LUAD. The expression of PPM1G was positively correlated with γδ­Τ cells, T helper 2 cells and natural killer CD56dim cells, and was negatively correlated with B cells, mast cells, plasmacytoid dendritic cells, T helper cells, macrophages, T cells, CD8 T cells, central memory T cells, effector memory T cells, neutrophils and T follicular helper cells. In addition, PPM1G was positively correlated with immune detection points. In conclusion, PPM1G may be involved in the control of the lung cancer cell cycle, and could be associated with prognosis and immune infiltration in patients with LUAD.

Developing a medium combination to attain similar glycosylation profile to originator by DoE and cluster analysis method.

Glycosylation is critical for monoclonal antibody production because of its impact on pharmacokinetics and pharmacodynamics. Modulation of glycan profile is frequently needed in biosimilar development. However, glycosylation profile is not a single value like that of cell culture titer, hence making it challenging for the Design of Experiment (DoE) methodology to be directly applied. In this study, a Her2-binding antibody was developed as a biosimilar to Herceptin. Cluster analysis was introduced to demonstrate the similarity of glycan profiles between the samples and the reference with specific value-distance. The glycosylation was subsequently optimized with the DoE method. Basal medium and feed medium were found to be the significant factors to the glycosylation pattern. Moreover, a combination of medium and feed strategy was developed to attain the most similar glycoprotein molecule to that of the originator biologic drug. This study may provide an additional option to evaluate multivariable factors and assess biosimilarity and/or comparability in monoclonal antibody production.

UHPLC-MS/MS method to determine FP-208 in human plasma and its application to a pharmacokinetic study.

Aim: FP-208 is a novel and effective small-molecule inhibitor blocking the mammalian target of rapamycin complex-1/mammalian target of rapamycin complex-2/PI3Ka. To investigate the pharmacokinetic profile of FP-208, a rapid and reliable analytical method was needed to be established to determine FP-208 in the plasma of patients with solid tumors. Materials & methods: FP208 was separated on a charged surface hybrid (CSH) C18 column (2.1 mm × 50 mm, 1.7 µm) after the plasma samples were purified using a protein precipitation method. Detection was performed on an AB Sciex 5500 mass spectrometer in the positive electrospray ionization mode. The established method was validated according to the bioanalytical guidelines. Conclusion: For the first time, the developed and validated method was successfully applied in the first-in-human study for FP-208 in patients with solid tumors after oral administration (Number: CTR20180683).

GroEL/ES mediated the in vivo recovery of TRAIL inclusion bodies in Escherichia coli.

Inclusion body (IB) formation generates substantial bio-waste in the pharmaceutical industry and remains a major challenge for heterologous protein expression. Although chaperones can be co-expressed to improve soluble protein yield, their contribution to IB processing in vivo has not been thoroughly studied. Here, a GroEL-GroES co-expressing strain and a deficient strain were constructed to study the in vivo recovery of recombinant human tumor necrosis factor-related apoptosis-inducing ligand (TRAIL). The interaction between GroEL/ES and TRAIL was simulated by molecular docking and identified by co-immunoprecipitation. The in vitro cytotoxicity of TRAIL IBs before and after in vivo recovery was subsequently determined by MTT assay. Additionally, IB structures were measured by Fourier transform infrared (FT-IR) spectroscopy and fluorescence spectroscopy. The results showed that after in vivo refolding, IBs retained lower levels of anti-tumor activity and fewer native-like ß-sheet structures. Fewer recoverable polypeptides were trapped in IBs after GroEL/ES co-expression and refolding in vivo. Therefore, GroEL/ES mediated the in vivo recovery of TRAIL IBs in Escherichia coli. These results may identify potential uses for IBs and provide additional insight into the detailed mechanisms of in vivo protein recovery.

Higher non-HDL-cholesterol to HDL-cholesterol ratio linked with increased nonalcoholic steatohepatitis.

BACKGROUND: Non-HDL-cholesterol to HDL-cholesterol (non-HDL-c/HDL-c) ratio is a feasible predictor for coronary heart disease, metabolic syndrome, and insulin resistance. Patients with nonalcoholic steatohepatitis (NASH) have an increased risk of developing cardiovascular problems and type 2 diabetes. However, the predictive role of non-HDL-c/HDL-c ratio in NASH hasn't been investigated yet. METHODS: We conducted a retrospective cohort study. A total of 3489 eligible subjects were selected in the present study. Prevalence and characteristics of NASH were demonstrated. Conditional logistic regression was used to analyze the association between non-HDL-c/HDL-c ratio and risks of NASH. Associations between non-HDL-c/HDL-c ratio and serum aminotransferase levels were also investigated. RESULTS: The overall prevalence of NASH was 6.13%, higher in male (6.89%) than that in female (5.04%). Interestingly, the prevalence of NASH showed a positive correlation with the elevation of non-HDL-c/HDL-c ratio (Pearson's Chi-squared test, linear trend 0.010, p <  0.05). The risk of NASH increased approximately 1.8-fold among subjects with higher non-HDL-c/HDL-c ratio. After adjustment for confounding factors, higher non-HDL-c/HDL-c ratio was still associated with a 54.4% increased risk of NASH. Male had higher risk of NASH than female when their non-HDL-c/HDL-c ratio increased. The risk of NASH in subjects with BMI more than 24 was 3 times higher than that in subjects with BMI less than 24. Every one unit increase in Non-HDL-c/HDL-c ratio was associated with 64.5% increase in ALT/AST level (p <  0.05) after adjustment for confounding factors. CONCLUSIONS: Our study provided strong evidence that subjects with higher non-HDL-c/HDL-c ratio had a higher risk of NASH, which suggested that non-HDL-c/HDL-c ratio might be a feasible predictor for NASH.

Enhanced production of soluble tumor necrosis factor-related apoptosis-inducing ligand in Escherichia coli using a novel self-cleavable tag system Fh8-ΔI-CM.

Escherichia coli is an essential host for large-scale expression of heterologous polypeptides. However, further applications are limited by the formation of potential protein aggregates. In this work, we developed a novel on-column tag removal and purification system based on Fh8 hydrophobic interaction chromatography purification and ΔI-CM self-cleavage to obtain soluble tumor necrosis factor-related apoptosis-inducing ligand (TRAIL). We evaluated several methods to improve TRAIL solubility and finally demonstrated that the Fh8 tag was a powerful solubility enhancer. Finally, we replaced the tobacco etch virus (TEV) protease site with a ΔI-CM self-cleavage intein to simplify the purification process. The released soluble TRAIL purity and yield reached 98.4% and 82.1 mg/L in shake flasks, respectively. Thus, the Fh8-ΔI-CM system enhanced target protein solubility by Fh8, enabled on-column tag removal and purification based on Fh8 calcium-binding properties and ΔI-CM self-cleavage properties, and promoted the release of highly active protein with high yield and purity. Overall, our findings suggest that this Fh8-ΔI-CM system could be used as a novel solubility-inducing and purification fusion tag for protein production in E. coli.

Metabolic engineering of Serratia marcescens MG1 for enhanced production of (3R)-acetoin.

BACKGROUND: Optically pure acetoin (AC) is an important platform chemical which has been widely used to synthesize novel optically active α-hydroxyketone derivatives and liquid crystal composites. RESULTS: In this study, slaC and gldA encoding meso-2,3-butanediol dehydrogenase (meso-2,3-BDH) and glycerol dehydrogenase (GDH), respectively, in S. marcescens MG1 were knocked out to block the conversion from AC to 2,3-butanediol (2,3-BD). The resulting strain MG14 was found to produce a large amount of optically pure (3R)-AC with a little 2,3-BD, indicating that another enzyme responsible for 2,3-BD formation except meso-2,3-BDH and GDH existed in the strain MG1. Furthermore, SlaR protein, a transcriptional activator of AC cluster, was overexpressed using PC promoter in the strain MG14, leading to enhancement of the (3R)-AC yield by 29.91%. The recombinant strain with overexpression of SlaR, designated as S. marcescens MG15, was used to perform medium optimization for improving (3R)-AC production. CONCLUSION: Under the optimized conditions, 39.91 ± 1.35 g/l (3R)-AC was produced by strain MG15 with the productivity of 1.11 g/l h and the conversion rate of 80.13%.

Berberine increases adipose triglyceride lipase in 3T3-L1 adipocytes through the AMPK pathway.

BACKGROUND: Obesity is closely related to the metabolism of triacylglycerol (TG) in adipocytes. Adipose triglyceride lipase (ATGL) and hormone-sensitive lipase (HSL) are rate-limiting enzymes that control the hydrolysis of TG. Effects on ATGL and HSL to increase lipolysis may counteract obesity. Berberine (BBR) is a compound derived from the Chinese medicine plant Coptis chinensis. In the present study we show the effects of BBR on ATGL and HSL and explore the potential underlying mechanisms of these effects. METHODS: The TG content in cells was measured using a colorimetric assay. The expressions of HSL, ATGL and GPAT3 were evaluated by Western-blotting. The expression of ATGL was also evaluated by real-time PCR and radioimmunoassay. Compound C, an inhibitor of AMP-activated protein kinase (AMPK), was used to explore the possible pathway that involved in the effect of BBR on ATGL. RESULTS: TG content of differentiated 3T3-L1 cells was significantly decreased by more than 10% after treated with BBR. In differentiated 3T3-L1 adipocytes, BBR increased the expression of p-HSL and ATGL, and these effects were time-depended (p <0.01). The effect of BBR on ATGL expression could be abolished by Compound C which suggested that AMPK pathway was involved in the effects of BBR on p-HSL and ATGL. CONCLUSIONS: BBR could increase the expression of ATGL and therefore stimulate basal lipolysis in mature adipocytes through the associated mechanisms related to the AMPK pathway.

Efficient refolding of the bifunctional therapeutic fusion protein VAS-TRAIL by a triple agent solution.

VAS-TRAIL is a bifunctional fusion protein that combines anti-angiogenic activity with tumor-selective apoptotic activity for enhanced anti-tumor efficacy. VAS-TRAIL is expressed as inclusion body in Escherichia coli, but protein refolding is difficult to achieve and results in low yields of bioactive protein. In this study, we describe an efficient method for VAS-TRAIL refolding. The solubilization of aggregated VAS-TRAIL was achieved by a triple agent solution, which consists of an alkaline solution (pH 11.5) containing 0.4M l-arginine and 2M urea. The solubilized protein showed high purity and preserved secondary structure according to fluorescence properties. VAS-TRAIL refolding was performed through stepwise dialysis and resulted in more than 50% recovery of the soluble protein. The function of l-arginine was additive with alkaline pH, as shown by the significant improvement in refolding yield (≈30%) by l-arginine-containing solubilization solutions compared with alkaline solubilization solutions without l-arginine. The refolded VAS-TRAIL also showed ß-sheet structures and the propensity for oligomerization. Bioassays showed that the refolded fusion protein exhibited the expected activities, including its apoptotic activities toward tumor and endothelial cells, which proposed its promising therapeutic potential.

A new open-loop fiber optic gyro error compensation method based on angular velocity error modeling.

With the open-loop fiber optic gyro (OFOG) model, output voltage and angular velocity can effectively compensate OFOG errors. However, the model cannot reflect the characteristics of OFOG errors well when it comes to pretty large dynamic angular velocities. This paper puts forward a modeling scheme with OFOG output voltage u and temperature T as the input variables and angular velocity error Δω as the output variable. Firstly, the angular velocity error Δω is extracted from OFOG output signals, and then the output voltage u, temperature T and angular velocity error Δω are used as the learning samples to train a Radial-Basis-Function (RBF) neural network model. Then the nonlinear mapping model over T, u and Δω is established and thus Δω can be calculated automatically to compensate OFOG errors according to T and u. The results of the experiments show that the established model can be used to compensate the nonlinear OFOG errors. The maximum, the minimum and the mean square error of OFOG angular velocity are decreased by 97.0%, 97.1% and 96.5% relative to their initial values, respectively. Compared with the direct modeling of gyro angular velocity, which we researched before, the experimental results of the compensating method proposed in this paper are further reduced by 1.6%, 1.4% and 1.42%, respectively, so the performance of this method is better than that of the direct modeling for gyro angular velocity.

Therapeutic effects of smecta or smectite powder on rats with paraquat toxication.

BACKGROUND: The plasma concentration of paraquat is closely related to the prognosis of patients with paraquat toxication, and the most common cause of death from paraquat poisoning is multiple organ failure (MOF). This study aimed to evaluate therapeutic effect of smecta on the plasma concentrations of paraquat and multi-organ injury induced by paraquat intoxication in rats. METHODS: A total of 76 healthy adult SD rats were randomly divided into group A (control group, n=6), group B (poisoned group, n=30) and group C (smecta-treated group, n=30). Rats in groups B and C were treated intragastrically with PQ at 50 mg/kg, and rats in group A was treated intragastrically with saline (1 mL). Rats in group C were given intragastrically smecta at 400 mg/kg 10 minutes after administration of PQ, while rats in other two groups were treated intragastrically with 1 mL saline at the same time. Live rats in groups B and C were sacrificed at 2, 6, 24, 48, 72 hours after administration of PQ for the determination of paraquat plasma concentrations and for HE staining of the lung, stomach and jejunum. The rats were executed at the end of trial by the same way in group A. RESULTS: The plasma concentration of paraquat (ng/mL) ranged from 440.314±49.776 to 4320.6150±413.947. Distinctive pathological changes were seen in the lung, stomach and jejunum in group B. Lung injuries deteriorated gradually, edema, leukocyte infiltration, pneumorrhagia, incrassated septa and lung consolidation were observed. Abruption of mucosa, hyperemic gastric mucosa and leukocyte infiltration were obvious in the stomach. The hemorrhage of jejunum mucosa, the abruption of villus, the gland damage with the addition of inflammatory cell infiltration were found. Compared to group B, the plasma concentration of paraquat reduced (P<0.01) and the pathological changes mentioned above were obviously alleviated in group C (P<0.05, P<0.01). CONCLUSION: Smecta reduced the plasma concentration of paraquat and alleviated pathologic injury of rats with PQ poisoning.

Involvement of guanylate cyclase and K+ channels in relaxation evoked by ferulate nitrate in rat aorta artery.

Vasorelaxant properties of N-2-(ferulamidoethyl)-nitrate (ferulate nitrate, FLNT), a newly synthesized nitrate, were compared with those of isosorbide dinitrate, nicorandil, nitroglycerin, and 8-bromoguanosine 3,5-cyclic monophosphate (8-Br-cGMP) in rat aorta pre-contracted by phenylephrine. FLNT produced vasorelaxation in a concentration-dependent manner (0.1 - 100 µM). The degree of relaxation induced by FLNT was similar to that induced by isosorbide dinitrate. In addition, removal of endothelium did not affect the relaxant effect of FLNT. FLNT caused a rightward shift of the cumulative concentration-response curves of phenylephrine and reduced the maximal efficacy of contraction. 1H-[1,2,4]Oxadiazolo-[4,3-a]quinoxalin-1-one (ODQ, 10 µM) and K(+)-channel blockers charybdotoxin (CHT, 0.1 µM) and BaCl(2) (1 µM) reduced the relaxant effect of FLNT in the endothelium-denuded arteries, whereas glibenclamide (1 µM) and 4-aminopyridine (1 mM) failed to influence FLNT-induced vasorelaxation. Furthermore, in the presence of ODQ, both CHT (0.1 µM) and BaCl(2) (1 µM) still significantly reduced the relaxation evoked by FLNT. Pretreatment of vessels with hydroxocobalamin, a nitric oxide scavenger, abolished the FLNT effect. These findings demonstrate that FLNT induces relaxation of the rat aorta rings endothelium-independently. Furthermore, we demonstrated that FLNT-induced vasorelaxation is related to its stimulation of soluble guanylate cyclase and activation of K(+) channels.

Simultaneous biohydrogen and bioethanol production from anaerobic fermentation with immobilized sludge.

The effects of organic loading rates (OLRs) on fermentative productions of hydrogen and ethanol were investigated in a continuous stirred tank reactor (CSTR) with attached sludge using molasses as substrate. The CSTR reactor with attached sludge was operated under different OLRs, ranging from 8 to 24 kg/m(3)·d. The H(2) and ethanol production rate essentially increased with increasing OLR. The highest H(2) production rate (10.74 mmol/h·L) and ethanol production rate (11.72 mmol/h·L) were obtained both operating at OLR = 24 kg/m(3)·d. Linear regression results show that ethanol production rate (y) and H(2) production rate (x) were proportionately correlated and can be expressed as y = 1.5365x - 5.054 (r(2) = 0.9751). The best energy generation rate was 19.08 kJ/h·L, which occurred at OLR = 24 kg/m(3)·d. In addition, the hydrogen yield was affected by the presence of ethanol and acetic acid in the liquid phase, and the maximum hydrogen production rate occurred while the ratio of ethanol to acetic acid was close to 1.

Ferilnic nirate produces delayed preconditioning against myocardial ischemia and reperfusion injury in rats.

To determine whether ferilnic nirate (FLNT) can precondition the rat heart against myocardial ischemia/reperfusion (I/R) damage and its mechanism, two groups of experiments were conducted. In the first group of experiments, rats were divided among four treatment groups: sham group; solvent with I/R (I/R control group); FLNT pretreatment with I/R (I/R FLNT group); and late ischemic preconditioning group (LPC group). In the second group of experiments without I/R, rats were divided into two treatment groups: control group and FLNT group. The results indicated that myocardial infarct size and the levels of creatine kinase and lactate dehydrogenase in the sera of the I/R FLNT group were significantly lower and the level of nitric oxide molecule and Mn-containing superoxide dismutase were significantly elevated in the heart tissue compared with I/R control group. The protein expression ratio of Bcl-2/Bax in heart tissue was significantly elevated in the I/R FLNT group. These results demonstrate FLNT is able to precondition rat hearts against myocardial ischemia/reperfusion damage to a similar level as that achieved via the late phase of ischemic preconditioning. The mechanism may involve the up-regulation of nitric oxide and the strengthening of anti-oxidant and anti-apoptosis cellular functions.