Recombinant therapeutic proteins degradation and overcoming strategies in CHO cells.

Mammalian cell lines are frequently used as the preferred host cells for producing recombinant therapeutic proteins (RTPs) having post-translational modified modification similar to those observed in proteins produced by human cells. Nowadays, most RTPs approved for marketing are produced in Chinese hamster ovary (CHO) cells. Recombinant therapeutic antibodies are among the most important and promising RTPs for biomedical applications. One of the issues that occurs during development of RTPs is their degradation, which caused by a variety of factors and reducing quality of RTPs. RTP degradation is especially concerning as they could result in reduced biological functions (antibody-dependent cellular cytotoxicity and complement-dependent cytotoxicity) and generate potentially immunogenic species. Therefore, the mechanisms underlying RTP degradation and strategies for avoiding degradation have regained an interest from academia and industry. In this review, we outline recent progress in this field, with a focus on factors that cause degradation during RTP production and the development of strategies for overcoming RTP degradation. KEY POINTS: • The recombinant therapeutic protein degradation in CHO cell systems is reviewed. • Enzymatic factors and non-enzymatic methods influence recombinant therapeutic protein degradation. • Reducing the degradation can improve the quality of recombinant therapeutic proteins.

A DFT study on methanol decomposition over single atom Pt/CeO2 catalysts: the effect of the position of Pt.

Pt/CeO2 catalysts exhibit excellent catalytic performance for the methanol dehydrogenation (MD) reaction. In this work, MD reactions on three systems of Pt1/CeO2(110)), Pt7/CeO2(110), and Pt1/Ce1-xO2(110) are investigated via density functional theory (DFT) calculations. The CH3OH adsorption, electronic structure of the catalyst, and mechanism of methanol decomposition (MD) are systematically calculated. The results reveal that the d-band center of the Pt atom moves away from the Fermi level in the order of Pt1/CeO2(110) < Pt7/CeO2(110) < Pt1/Ce1-xO2(110), and the order of the activity of the MD reaction is Pt1/CeO2(110) < Pt7/CeO2(110) < Pt1/Ce1-xO2(110). The results of the microkinetic dynamics simulation verify that only Pt1/Ce1-xO2(110) is conducive to the decomposition of methanol at low temperatures (373 K), and the products CO and H2 are easily dissociated from the catalyst surface. This work uncovers that both the small size and the Ce vacancy substituted sites of Pt favor the performance of the Pt/CeO2 catalyst, and provides theoretical guidance for the construction and design of efficient metal-support catalysts for the MD reaction.

Efficacy of Zidovudine-Amikacin Combination Therapy In Vitro and in a Rat Tissue Cage Infection Model against Amikacin-Resistant, Multidrug-Resistant Enterobacteriales.

Multidrug-resistant (MDR) Enterobacteriales infections have become an urgent global threat to public health. The aim of this study was to evaluate the efficacy of zidovudine-amikacin combination therapy in vitro and in vivo. Molecular characteristics and antibiotic resistance profiles of 53 amikacin-resistant MDR, extensively drug-resistant (XDR), or pan-drug-resistant (PDR) clinical isolates were examined via PCR and susceptibility testing. Checkerboard assays were performed for these 53 isolates to assess in vitro synergistic effects of the zidovudine-amikacin combination, and static time-kill experiments were performed for four XDR or PDR Enterobacteriales isolates. A Galleria mellonella model and a rat tissue cage infection model were established to assess in vivo synergistic effects. The aac(6')-Ib gene was detected in 25 (47.2%) isolates, followed by armA in 5 (9.4%) isolates, rmtB in 27 (50.9%) isolates, and rmtC in 3 (5.8%) isolates. Checkerboard assays showed the synergy of this combination against 38 (71.7%) isolates. The time-kill assays further confirmed that zidovudine strongly synergized with amikacin against four XDR or PDR Enterobacteriales isolates. The Galleria mellonella model study showed that the survival benefit of zidovudine-amikacin combination therapy was significantly better than that of monotherapy for those four Enterobacteriales isolates. Furthermore, the rat tissue cage infection model study showed that zidovudine-amikacin combination therapy displayed more potent bactericidal activity than monotherapy after 3 and 7 days of treatment for the above four isolates. Our data support the idea that the zidovudine-amikacin combination could be a plausible alternative therapy against infections with amikacin-resistant MDR Enterobacteriales, especially with XDR and PDR Enterobacteriales. IMPORTANCE Our study revealed for the first time that the zidovudine-amikacin combination shows a significant bactericidal effect against amikacin-resistant MDR, XDR, and PDR Enterobacteriales. Second, using in vitro and in vivo approaches, our study showed that zidovudine strongly synergized with amikacin against amikacin-resistant MDR Enterobacteriales isolates. Most importantly, with regard to survival benefit, pharmacokinetics, and bactericidal effects, our in vivo experiment demonstrated the effectiveness of zidovudine-amikacin.

Research on traceability strategy of food supply chain considering delay effect.

The traceability system has significantly contributed to ensure food safety and quality. However, the biggest difficulty in food traceability is the numerous links from field to table, and there is no stable strategic partnership between supply chain members and the lack of social responsibility of some practitioners. Thus, this study aims to seek the best traceability strategy for companies in centralized model and decentralized model, respectively. Therefore, we have constructed a differential game model based on the delay effect to determine the optimal traceability level and traceable goodwill and compare the profits of the food supply chain (FSC). The results show that the delay time is positively related to the level of traceability effort and has a high impact on the traceable goodwill. Companies in the FSC can formulate optimal traceability strategies based on delay time and foster improvement in food safety and quality.

Effect of point defects on acetylene hydrogenation reaction over Ni(111) surface: a density functional theory study.

Density functional theory (DFT) calculations are carried out to investigate the effect of point defects on acetylene hydrogenation reaction over Ni(111) surface with three different defect concentrations (DC = 0.0500, 0.0625, and 0.0833), compared with the perfect Ni(111) surface. The adsorptions of C2 species and H atoms and the mechanism of acetylene hydrogenation via the ethylene pathway are systematically analyzed. The results indicate that the existence of defects will make C2 species and H atoms more inclined to adsorb near the defects. Introducing an appropriate amount of point defect concentration can enhance the catalytic activity and ethylene selectivity of Ni. In this work, DC = 0.0625 Ni(111) surface has the highest catalytic activity and selectivity of ethylene. This work provides useful theoretical information on the effect of defects on acetylene hydrogenation and is helpful for the design of Ni and related metal catalysts with defects.

Hepatic paraoxonase 1 ameliorates dysfunctional high-density lipoprotein and atherosclerosis in scavenger receptor class B type I deficient mice.

BACKGROUND: High-density lipoprotein (HDL) plays an antiatherogenic role by mediating reverse cholesterol transport (RCT), antioxidation, anti-inflammation, and endothelial cell protection. Recently, series of evidence have shown that HDL can also convert to proatherogenic HDL under certain circumstances. Plasma paraoxonase 1 (PON1) as an HDL-bound esterase, is responsible for most of the antioxidant properties of HDL. However, whether PON1 can serve as a therapeutic target of dysfunctional HDL-related atherosclerosis remains unclear. METHODS: In this study, scavenger receptor class B type I deficient (Scarb1-/- ) mice were used as the animal model with dysfunctional HDL and increased atherosclerotic susceptibility. Hepatic PON1 overexpression and secretion into circulation were achieved by lentivirus injection through the tail vein. We monitored plasma lipids levels and lipoprotein profiles in Scarb1-/- mice, and measured the levels and activities of proteins associated with HDL function. Meanwhile, lipid deposition in the liver and atherosclerotic lesions was quantified. Hepatic genes relevant to HDL metabolism and inflammation were analyzed. RESULTS: The results showed the relative levels of PON1 in liver and plasma were increased by 1.1-fold and 1.6-fold, respectively, and mean plasma PON1 activity was increased by 63%. High-level PON1 increased the antioxidative and anti-inflammatory properties, promoted HDL maturation and macrophage cholesterol efflux through increasing HDL functional proteins components apolipoprotein A1 (APOA1), apolipoprotein E (APOE), and lecithin-cholesterol acyltransferase (LCAT), while decreased inflammatory protein markers, such as serum amyloid A (SAA), apolipoprotein A4 (APOA4) and alpha 1 antitrypsin (A1AT). Furthermore, hepatic PON1 overexpression linked the effects of antioxidation and anti-inflammation with HDL metabolism regulation mainly through up-regulating liver X receptor alpha (LXRα) and its downstream genes. The pleiotropic effects involved promoting HDL biogenesis by raising the level of APOA1, increasing cholesterol uptake by the liver through the APOE-low density lipoprotein receptor (LDLR) pathway, and increasing cholesterol excretion into the bile, thereby reducing hepatic steatosis and aorta atherosclerosis in Western diet-fed mice. CONCLUSIONS: Our study reveals that high-level PON1 improved dysfunctional HDL and alleviated the development of atherosclerosis in Scarb1-/- mice. It is suggested that PON1 represents a promising target of HDL-based therapeutic strategy for HDL-related atherosclerotic cardiovascular disease.

Anion exchange behavior of MIIAl layered double hydroxides: a molecular dynamics and DFT study.

The ion exchange reaction has been extensively used in the field of synthesis of functionalized supramolecular materials such as layered double hydroxides (LDHs), ion-embedded batteries, sewage disposal and so on. In this work, the factors influencing the anion exchange behavior in the LDH gallery, such as the exchange domain, the exchange order, the driving force, and the diffusion of the anions, are investigated systematically using molecular dynamics (MD) simulations and density functional theory (DFT) methods in view of both thermodynamics and dynamics. 159 models of MIIRAl-A-LDHs (MII = Mg, Ni, Zn; R = 1.4-8, A = OH-, Cl-, Br-, NO3-, HCOO-, C6H5SO3-, CO32-, SO42-, and PO43-, respectively) are calculated. The results reveal that the anion exchange domain (interlayer distance) in LDHs is determined not only by the size and their arrangement modes of the guest anions, but also by the charges the anions carry. The relative binding energies of different anions and the Gibbs free energy changes of the anion exchange reactions in LDHs decrease in the order of PO43- > CO32- > SO42- > OH- > Cl- > Br- > HCOO- > NO3- > C6H5SO3-, which is in accordance with the experimental anion exchange order. The stronger the hydrogen bonding between the anion and the host, the larger the charge transfer, and the smaller the electronegativity of the anion, the more difficult it is for the anion to be exchanged out from LDH interlayer. In addition, for the anions with the same charges, the relative binding energy is linearly well correlated with the interlayer spacing. By analyzing the contribution of each energetic item comprising the total potential energy, it is found that the major driving force of anion exchange is the electrostatic force. The diffusion coefficient (D) along the c direction is nearly equal to zero, suggesting that the diffusion of anions occurs mainly in the ab plane of the LDH cell. It also can be inferred that when the cell parameter c < 24.0 Å, the anion exchange order is mainly determined by the thermodynamic factors, whereas when c > 24.0 Å, both the thermodynamic and the dynamic factors cast the same effect on the anion exchange behavior. This work provides an in-depth understanding of the anion exchange behavior, and is helpful guidance for the design and synthesis of functionalized guest anion intercalated LDHs and related materials using the anion-exchange method.

DFT study on MgAl-layered double hydroxides with different interlayer anions: structure, anion exchange, host-guest interaction and basic sites.

The guest anions play a key role in the construction of layered double hydroxide (LDH)-based host-guest functional materials. In this work, the orientation of the interlayer species, interlayer distances, binding energies, electronic density differences and density of states of the MgnAl-LDHs (n = 1.6, 2.0, 2.6, 3.5, 5.0, and 8.0) with nine different anions (F-, Cl-, Br-, I-, OH-, NO3-, CO32-, SO42-, and PO43-) are calculated by density functional theory (DFT). The results reveal that the LDHs containing the anions with more inclined arrangement, larger size, lower charge and with a larger number of interlayer water molecules show larger interlayer distances. The higher anion charge leads to a larger binding energy for LDHs, and the order of binding energy implies that the sequence of anion exchange is PO43- > CO32- > SO42- > OH- > F- > Cl- > Br- > NO3- > I-. The interactions between interlayer species and the host layer or the interlayer water molecules are mainly derived from the electrostatic interactions. The main components of the valence band maximum (VBM) and conduction band minimum (CBM) of MgAl-LDHs are derived from p orbitals of halogen anions or the O-2p orbitals of other anions, and the Mg-2p orbital, respectively. This illustrates that the most basic sites of MgAl-LDHs are the interlayer anions rather than the hydroxyl group in the layer, while the most acidic sites are Mg in the layers. And LDHs containing anions with higher charge show stronger basicity. The calculation results agree well with the experimental findings. This work provides effective theoretical information for the design and preparation of the anion-controlled functional LDHs or related materials with prospective applications.

Establishment and evaluation of a novel mouse model of peri/postmenopausal depression.

Women are believed to be more vulnerable to develop depressive symptoms during the perimenopause compared to postmenopause. The traditional bilateral ovariectomy and chronic mild stress (CMS) stimulation animal model produces a postmenopausal depressive-like state but the transition from perimenopausal period to postmenopausal period was ignored. Thus we establish a novel animal model in which the mice were stimulated by CMS for three months and removed the ovaries by two-step operation, and then evaluate whether this novel model could be much better for preclinical study used as a peri/postmenopause depressive model. The present study systemically evaluated the changes induced by two-step ovariectomy plus CMS in the mice. The depression-like behaviors, the levels of corticosterone, estrogen, pro-inflammatory factors, neurotransmitters, as well as brain-derived neurotrophic factor were determined; the changes of estrogen receptors, serotonin receptors, uterine weight and bone microarchitecture were also observed. The results show that the behaviors and biochemical indexes of mice changed gradually over time. Our study suggests that this two-step ovariectomy operation plus CMS successfully establishes a more reasonable peri/postmenopausal depression animal model which effectively simulates the clinical symptoms of peri/postmenopausal depressive women.

Antagonizing peroxisome proliferator-activated receptor γ facilitates M1-to-M2 shift of microglia by enhancing autophagy via the LKB1-AMPK signaling pathway.

Microglia-mediated neuroinflammation plays a dual role in various brain diseases due to distinct microglial phenotypes, including deleterious M1 and neuroprotective M2. There is growing evidence that the peroxisome proliferator-activated receptor γ (PPARγ) agonist rosiglitazone prevents lipopolysaccharide (LPS)-induced microglial activation. Here, we observed that antagonizing PPARγ promoted LPS-stimulated changes in polarization from the M1 to the M2 phenotype in primary microglia. PPARγ antagonist T0070907 increased the expression of M2 markers, including CD206, IL-4, IGF-1, TGF-ß1, TGF-ß2, TGF-ß3, G-CSF, and GM-CSF, and reduced the expression of M1 markers, such as CD86, Cox-2, iNOS, IL-1ß, IL-6, TNF-α, IFN-γ, and CCL2, thereby inhibiting NFκB-IKKß activation. Moreover, antagonizing PPARγ promoted microglial autophagy, as indicated by the downregulation of P62 and the upregulation of Beclin1, Atg5, and LC3-II/LC3-I, thereby enhancing the formation of autophagosomes and their degradation by lysosomes in microglia. Furthermore, we found that an increase in LKB1-STRAD-MO25 complex formation enhances autophagy. The LKB1 inhibitor radicicol or knocking down LKB1 prevented autophagy improvement and the M1-to-M2 phenotype shift by T0070907. Simultaneously, we found that knocking down PPARγ in BV2 microglial cells also activated LKB1-AMPK signaling and inhibited NFκB-IKKß activation, which are similar to the effects of antagonizing PPARγ. Taken together, our findings demonstrate that antagonizing PPARγ promotes the M1-to-M2 phenotypic shift in LPS-induced microglia, which might be due to improved autophagy via the activation of the LKB1-AMPK signaling pathway.

Rosiglitazone Exerts an Anti-depressive Effect in Unpredictable Chronic Mild-Stress-Induced Depressive Mice by Maintaining Essential Neuron Autophagy and Inhibiting Excessive Astrocytic Apoptosis.

There is increasing interest in the association between depression and the development of metabolic diseases. Rosiglitazone, a therapeutic drug used to treat type 2 diabetes mellitus, has shown neuroprotective effects in patients with stroke and Alzheimer's disease. The present study was performed to evaluate the possible roles of rosiglitazone in in vivo (unpredictable chronic mild stress-induced depressive mouse model) and in vitro (corticosterone-induced cellular model) depressive models. The results showed that rosiglitazone reversed depressive behaviors in mice, as indicated by the forced swimming test and open field test. Rosiglitazone was also found to inhibit the inflammatory response, decrease corticosterone levels, and promote astrocyte proliferation and neuronal axon plasticity in the prefrontal cortex of mice. This series of in vivo and in vitro experiments showed that autophagy among neurons was inhibited in depressive models and that rosiglitazone promoted autophagy by upregulating LKB1, which exerted neuroprotective effects. Rosiglitazone was also found to activate the Akt/CREB pathway by increasing IGF-1R expression and IGF-1 protein levels, thereby playing an anti-apoptotic role in astrocytes. Rosiglitazone's autophagy promotion and neuroprotective effects were found to be reversed by the PPARγ antagonist T0070907 in primary neurons and by PPARγ knockdown in an N2a cell line. In conclusion, we found that rosiglitazone protects both neurons and astrocytes in in vivo and in vitro depressive models, thereby playing an anti-depressive role. These findings suggest that PPARγ could be a new target in the development of anti-depressive drugs.

Activation of ATP-sensitive potassium channel by iptakalim normalizes stress-induced HPA axis disorder and depressive behaviour by alleviating inflammation and oxidative stress in mouse hypothalamus.

Stress-induced disturbance of the hypothalamic-pituitary-adrenal (HPA) axis is strongly implicated in incidence of mood disorders. A heightened neuroinflammatory response and oxidative stress play a fundamental role in the dysfunction of the HPA axis. We have previously demonstrated that iptakalim (Ipt), a new ATP-sensitive potassium (K-ATP) channel opener, could prevent oxidative injury and neuroinflammation against multiple stimuli-induced brain injury. The present study was to demonstrate the impacts of Ipt in stress-induced HPA axis disorder and depressive behavior. We employed 2 stress paradigms: 8 weeks of continuous restraint stress (chronic restraint stress, CRS) and 2h of restraint stress (acute restraint stress, ARS), to mimic both chronic stress and severe acute stress. Prolonged (4 weeks) and short-term (a single injection) Ipt treatment was administered 30min before each stress paradigm. We found that HPA axis was altered after stress, with different responses to CRS (lower ACTH and CORT, higher AVP, but normal CRH) and ARS (higher CRH, ACTH and CORT, but normal AVP). Both prolonged and short-term Ipt treatment normalized stress-induced HPA axis disorders and abnormal behaviors in mice. CRS and ARS up-regulated mRNA levels of inflammation-related molecules (TNFα, IL-1ß, IL-6 and TLR4) and oxidative stress molecules (gp91phox, iNOS and Nrf2) in the mouse hypothalamus. Double immunofluorescence showed CRS and ARS increased microglia activation (CD11b and TNFα) and oxidative stress in neurons (NeuN and gp91phox), which were alleviated by Ipt. Therefore, the present study reveals that Ipt could prevent against stress-induced HPA axis disorders and depressive behavior by alleviating inflammation and oxidative stress in the hypothalamus.

High-density genetic linkage map construction and QTL mapping of grain shape and size in the wheat population Yanda1817 × Beinong6.

High-density genetic linkage maps are necessary for precisely mapping quantitative trait loci (QTLs) controlling grain shape and size in wheat. By applying the Infinium iSelect 9K SNP assay, we have constructed a high-density genetic linkage map with 269 F 8 recombinant inbred lines (RILs) developed between a Chinese cornerstone wheat breeding parental line Yanda1817 and a high-yielding line Beinong6. The map contains 2431 SNPs and 128 SSR & EST-SSR markers in a total coverage of 3213.2 cM with an average interval of 1.26 cM per marker. Eighty-eight QTLs for thousand-grain weight (TGW), grain length (GL), grain width (GW) and grain thickness (GT) were detected in nine ecological environments (Beijing, Shijiazhuang and Kaifeng) during five years between 2010-2014 by inclusive composite interval mapping (ICIM) (LOD ≥ 2.5). Among which, 17 QTLs for TGW were mapped on chromosomes 1A, 1B, 2A, 2B, 3A, 3B, 3D, 4A, 4D, 5A, 5B and 6B with phenotypic variations ranging from 2.62% to 12.08%. Four stable QTLs for TGW could be detected in five and seven environments, respectively. Thirty-two QTLs for GL were mapped on chromosomes 1B, 1D, 2A, 2B, 2D, 3B, 3D, 4A, 4B, 4D, 5A, 5B, 6B, 7A and 7B, with phenotypic variations ranging from 2.62% to 44.39%. QGl.cau-2A.2 can be detected in all the environments with the largest phenotypic variations, indicating that it is a major and stable QTL. For GW, 12 QTLs were identified with phenotypic variations range from 3.69% to 12.30%. We found 27 QTLs for GT with phenotypic variations ranged from 2.55% to 36.42%. In particular, QTL QGt.cau-5A.1 with phenotypic variations of 6.82-23.59% was detected in all the nine environments. Moreover, pleiotropic effects were detected for several QTL loci responsible for grain shape and size that could serve as target regions for fine mapping and marker assisted selection in wheat breeding programs.

[Pathological characteristics of sudden death caused by coronary thrombosis].

OBJECTIVE: To explore medico-legal characteristics of sudden death caused by coronary heart disease combined with coronary thrombosis. METHODS: Ninety-six cases of sudden death caused by coronary heart disease were collected and divided into two groups: thrombus positive and thrombus negative groups. The time onset, induction and pathological features of coronary artery disease were analyzed. RESULTS: Two groups showed man-dominant population. There were no statistical significant differences in season, circadian rhythm and induction factor. The thrombus positive group (age < 40) showed a higher disease incidence. Heart weight and degree of coronary stenosis were lower in thrombus positive group. However, there was no statistical difference in the number of atherosclerotic coronary arteries (> or = 2), the length of coronary lesions and myocardial infarct. But thrombosis positive group showed lower tendency. CONCLUSION: Two groups are man-dominant population and similar induction factor, lesion position, mechanism of death. But thrombus positive group appeared more in a younger population and the degree of coronary stenosis is milder than thrombus negative group. Forensic pathologists should pay more attention to these characteristics in death investigation.

[Greenhouse gas emission from reservoir and its influence factors].

Reservoirs are significant sources of emissions of the greenhouse gases. Discussing greenhouse gas emission from the reservoirs and its influence factors are propitious to evaluate emission of the greenhouse gas accurately, reduce gas emission under hydraulic engineering and hydropower development. This paper expatiates the mechanism of the greenhouse gas production, sums three approaches of the greenhouse gas emission, which are emissions from nature emission of the reservoirs, turbines and spillways and downstream of the dam, respectively. Effects of greenhouse gas emission were discussed from character of the reservoirs, climate, pH of the water, vegetation growing in the reservoirs and so on. Finally, it has analyzed the heterogeneity of the greenhouse gas emission as well as the root of the uncertainty and carried on the forecast with emphasis to the next research.