Expression,purification, and breast cancer cell inhibiting effect of recombinant human lactoferrin C-lobe

Lactoferrin (LTF), a multifunctional glycoprotein of the transferrin family mainly found in exotic secretions in mammals, is an important defense molecule against not only microbial invasion but also tumors. It folds into two globular domains (N- and C-lobes) each containing an iron-binding site. The cationic antimicrobial peptide in N-lobe is known to exert anti-tumor effect via a non-receptor-mediated pathway. However, whether LTF C-lobe also contributes to its anti-tumor activity remains to be investigated. In this study, a human LTF fragment (amino acid residues 343–682) covering the C-lobe was expressed with a histidine tag in E. coli and the purified polypeptide refolded through a series of buffer changing procedure. The resultant recombinant protein caused significant growth arrest of breast carcinoma cells MDA-MB-231 in a dose- and time-dependent manner, evidently via induction of apoptosis of the cell. Our data suggest a positive role for the C-lobe of human LTF in controlling tumors in vitro.     

Macrophage migration inhibitory factor in the regulation of myoblast proliferation and differentiation

Obesity is documented to be a state of chronic mild inflammation associated with increased macrophage infiltration into adipose tissue and liver and skeletal muscle. As a pleiotropic inflammatory mediator, macrophage migration inhibitory factor (MIF) is associated with metabolic disease, so MIF may signal molecular links between adipocytes and myocytes. MIF expression was modified during myoblast differentiation, but the role of MIF during this process is unclear. C2C12 cells were transfected with MIF to investigate their role during differentiation. MIF expression attenuated C2C12 differentiation. It did not change proliferation, but downregulated cyclin D1 and CDK4, causing cell accumulation in the G1 phase. p21 protein was increased significantly and MyoD, MyoG, and p21 mRNA also increased significantly in the C2C12 cells treated with ISO-1, suggesting that inhibition of MIF promotes differentiation. MIF inhibits the myoblast differentiation by affecting the cell cycle progression, but does not affect proliferation.     

Wasabi leaf extracts attenuate adipocyte hypertrophy through PPARγ and AMPK

Hypertrophy of adipocytes in obese adipose tissues causes metabolic abnormality by adipocytokine dysregulation, which promotes type 2 diabetes mellitus, hypertension, and dyslipidemia. We investigated the effects of wasabi (Wasabia japonica Matsum) leaf extracts on metabolic abnormalities in SHRSP.Z-Leprfa/IzmDmcr rats (SHRSP/ZF), which are a model of metabolic syndrome. Male SHRSP/ZF rats aged 7 weeks were divided into two groups: control and wasabi leaf extract (WLE) groups, which received water or oral treatment with 4 g/kg/day WLE for 6 weeks. WLE improved the body weight gain and high blood pressure in SHRSP/ZF rats, and the plasma triglyceride levels were significantly lower in the WLE group. Adipocyte hypertrophy was markedly prevented in adipose tissue. The expression of PPARγ and subsequent downstream genes was suppressed in the WLE group adipose tissues. Our data suggest that WLE inhibits adipose hypertrophy by suppressing PPARγ expression in adipose tissue and stimulating the AMPK activity by increased adiponectin.     

Combined metabolomics and proteomics reveals hypoxia as a cause of lower productivity on scale‐up to a 5000‐liter CHO bioprocess

Large‐scale bioprocessing is key to the successful manufacturing of a biopharmaceutical. However, cell viability and productivity are often lower in the scale‐up from laboratory to production. In this study, we analyzed CHO cells, which showed lower percent viabilities and productivity in a 5‐KL production scale bioreactor compared to a 20‐L bench‐top scale under seemingly identical process parameters. An increase in copper concentration in the media from 0.02 µM to 0.4 µM led to a doubling of percent viability in the production scale albeit still at a lower level than the bench‐top scale. Combined metabolomics and proteomics revealed the increased copper reduced the presence of reactive oxygen species (ROS) in the 5‐KL scale process. The reduction in oxidative stress was supported by the increased level of glutathione peroxidase in the lower copper level condition. The excess ROS was shown to be due to hypoxia (intermittent), as evidenced by the reduction in fibronectin with increased copper. The 20‐L scale showed much less hypoxia and thus less excess ROS generation, resulting in little to no impact to productivity with the increased copper in the media. The study illustrates the power of 'Omics in aiding in the understanding of biological processes in biopharmaceutical production.     

Self-preserving lognormal volume-size distributions of starch granules in developing sweetpotatoes and modulation of their scale parameters by a starch synthase II (SSII)

Main conclusion

Starch granule size distributions in plant tissues, when determined in high resolution and specifiedproperly as a frequency function, could provide useful information on the granule formation and growth.

Abstract

To better understand genetic control of physical properties of starch granules, we attempted a new approach to analyze developmental and genotypic effects on morphology and size distributions of starch granules in sweetpotato storage roots. Starch granules in sweetpotatoes exhibited low sphericity, many shapes that appeared to be independent of genotypes or developmental stages, and non-randomly distributed sizes. Granule size distributions of sweetpotato starches were determined in high resolution as differential volume-percentage distributions of volume-equivalent spherical diameters, rigorously curve-fitted to be lognormal, and specified using their geometric means \(\bar{x}^{*}\) and multiplicative standard deviations \(s^{*}\) in a \(\bar{x}^{*} \times /({\text{multiply/divide}})s^{*}\) form. The scale (\(\bar{x}^{*}\)) and shape (\(\bar{s}^{*}\)) of these distributions were independently variable, ranging from 14.02 to 19.36 μm and 1.403 to 1.567, respectively, among 22 cultivars/clones. The shape (\(s^{*}\)) of granule lognormal volume-size distributions of sweetpotato starch were found to be highly significantly and inversely correlated with their apparent amylose contents. More importantly, granule lognormal volume-size distributions of starches in developing sweetpotatoes displayed the same self-preserving kinetics, i.e., preserving the shape but shifting upward the scale, as those of particles undergoing agglomeration, which strongly indicated involvement of agglomeration in the formation and growth of starch granules. Furthermore, QTL analysis of a segregating null allele at one of three homoeologous starch synthase II loci in a reciprocal-cross population, which was identified through profiling starch granule-bound proteins in sweetpotatoes of diverse genotypes, showed that the locus is a QTL modulating the scale of granule volume-size distributions of starch in sweetpotatoes.

     

Life Cycle Energy and Greenhouse Gas Emissions of Automobiles Using Aluminum in China

Transportation is a major part of energy consumption and greenhouse gas (GHG) emissions. Aluminum (Al) as a light metal can reduce vehicle weight, energy consumption, and pollutant emissions, but Al production is energy intensive. The main contents of this study are the following: (1) create the life cycle inventory of Al parts based on the energy background in China and (2) evaluate the energy savings and GHG reduction for the vehicle when steel parts are replaced by Al parts. Although there is a considerable reduction in energy consumption of per tonne Al in China owing to continuing development of process technology in recent years, energy consumption is higher than the world average level and European level. Over the vehicle's life cycle driving of 200,000 kilometers, the vehicle was found to avoid 1,447 to 1,590 liters of gasoline consumption when six typical steel parts were replaced by Al parts. Based on the current technology, the breakeven distance was calculated, resulting in a net energy benefit to use the lightweight Al parts compared with steel parts. A sensitivity analysis was conducted to show different energy savings by considering secondary weight reduction and different driving distance. The results indicate that weight reduction by using Al is quite effective to reduce the energy consumption and GHG of transportation.     

A Cocktail ELISA Semi‐nested RT‐PCR Assay to Detect Bean pod mottle virus in Soya bean Seeds

Bean pod mottle virus (BPMV) has been identified as an important pathogen for plant quarantine in China because large quantities of soya bean seeds (approximately 7 × 10⁷ tons) are imported annually. To develop a practical detection programme for BPMV, a cocktail enzyme‐linked immunosorbent assay (ELISA) nested RT‐PCR using a combination of serological and molecular methods was designed for soya bean seeds. The single‐vessel detection assay was performed in a 96‐well ELISA plate, which served as a carrier for the subsequent nested RT‐PCR assay. Assay specificity was demonstrated by the production of the expected 330‐ and 296‐bp bands using the external and internal primers, respectively. This method was 10⁴‐fold more sensitive than immunocapture‐RT‐PCR (IC‐RT‐PCR). In particular, it is important to note that this assay resulted in successful micro‐extraction from soya bean seeds and combined the advantages of each individual technique. The cocktail ELISA nested RT‐PCR is a specific, sensitive, rapid and economical procedure to rapidly identify and characterize BPMV and could be suitable for both primary‐level platforms and laboratories.     

Exchange of the VP5 of infectious bursal disease virus in a serotype I strain with that of a serotype II strain reduced the viral replication and cytotoxicity

Infectious bursal disease virus (IBDV), belonging to Avibirnavirus genus in the Birnaviridae family, consists of two segments of double-strand RNA. There are two distinct serotypes of IBDV, the pathogenic serotype I and the non-pathogenic serotype II. Comparison of the deduced amino acid sequences of a panel of VP5 genes retrieved from GenBank revealed a high identity among strains within the serotype I or serotype II group but a low identity between strains across two serotypes. In this study, we rescued two mosaic viruses, rGtGxVP5 and rGt2382VP5 by exchanging the VP5 gene of a cell culture-adapted serotype I Gt strain with its counterpart of the very virulent IBDV Gx strain, or a non-pathogenic 23/82 strain of the serotype II. In comparison to the parental strain rGt virus, the rGtGxVP5 showed the similar viral replication, cytotoxicity and the ability of inducing apoptosis; however, the other mosaic virus rGt2382VP5 had a lower titer and a reduced cytotoxicity. Although exchange of VP5 within serotype I group did not alter the viral replication and cytotoxicity of Gt strain, exchange of VP5 in the serotype I with that of a serotype II reduced the viral replication and cytotoxicity on chicken embryo fibroblast (CEF) cells. Therefore, the VP5 of serotype II may be one of the factors responsible for the distinct pathogenic features of two serotypes.