Stable high volumetric production of glycosylated human recombinant IFNalpha2b in HEK293 cells

Background

Mammalian cells are becoming the prevailing expression system for the production of recombinant proteins because of their capacity for proper protein folding, assembly, and post-translational modifications. These systems currently allow high volumetric production of monoclonal recombinant antibodies in the range of grams per litre. However their use for large-scale expression of cytokines typically results in much lower volumetric productivity.

Results

We have engineered a HEK293 cell clone for high level production of human recombinant glycosylated IFNα2b and developed a rapid and efficient method for its purification. This clone steadily produces more than 200 mg (up to 333 mg) of human recombinant IFNα2b per liter of serum-free culture, which can be purified by a single-step cation-exchange chromatography following media acidification and clarification. This rapid procedure yields 98% pure IFNα2b with a recovery greater than 70%. Purified IFNα2b migrates on SDS-PAGE as two species, a major 21 kDa band and a minor 19 kDa band. N-terminal sequences of both forms are identical and correspond to the expected mature protein. Purified IFNα2b elutes at neutral pH as a single peak with an apparent molecular weight of 44,000 Da as determined by size-exclusion chromatography. The presence of intramolecular and absence of intermolecular disulfide bridges is evidenced by the fact that non-reduced IFNα2b has a greater electrophoretic mobility than the reduced form. Treatment of purified IFNα2b with neuraminidase followed by O-glycosidase both increases electrophoretic mobility, indicating the presence of sialylated O-linked glycan. A detailed analysis of glycosylation by mass spectroscopy identifies disialylated and monosialylated forms as the major constituents of purified IFNα2b. Electron transfer dissociation (ETD) shows that the glycans are linked to the expected threonine at position 106. Other minor glycosylated forms and non-sialylated species are also detected, similar to IFNα2b produced naturally by lymphocytes. Further, the HEK293-produced IFNα2b is biologically active as shown with reporter gene and antiviral assays.

Conclusion

These results show that the HEK293 cell line is an efficient and valuable host for the production of biologically active and glycosylated human IFNα2b.     

Food Inequality Negatively Impacts Cardiac Health in Rabbits

Background

Individuals with lower socioeconomic status experience higher rates of mortality and are more likely to suffer from numerous diseases. While some studies indicate that humans who suffer from social inequality suffer generally worse health, to our knowledge no controlled experiments of this nature have been done in any species. Lipofuscin is a highly oxidized cross-linked aggregate consisting of oxidized protein and lipid clusters. This eminent terminal oxidation outcome accumulates within cells during aging process.

Methodology/Principal Findings

Thirty two rabbits were assigned into four groups randomly of eight each. The first group encountered food deprivation for eight weeks and was kept in an isolated situation. The second group was food deprived for eight weeks but encountered to other groups continuously. The third group suffered two weeks of deprivation and then received free access to food. The fourth group had free access to diet without any deprivation. All hearts were removed for histopathological evaluation. Cross-sections of hearts were examined by light microscopy for the presence of yellow-brown Lpofuscin pigment granules. Here we show that relative food deprivation can cause accumulation of Lipofuscin pigmentation. We find that cardiac Lipofuscin deposition increases the most in the inequitable condition in which food deprived individuals observe well-fed individuals.

Conclusions/Significance

Our findings demonstrate that a sense of inequality in food intake can promote aging more than food deprivation alone. These findings should be considered as a basis for further studies on the physiological mechanisms by which inequality negatively impacts health and well-being.     

DNA damage induces two distinct modes of cell death in ovarian carcinomas

Activation of p53 by cellular stress may lead to either cell cycle arrest or apoptotic cell death. Restrictions in a cell's ability to halt the cell cycle might, in turn, cause mitotic catastrophe, a delayed type of cell death with distinct morphological features. Here, we have investigated the contribution of p53 and caspase-2 to apoptotic cell death and mitotic catastrophe in cisplatin-treated ovarian carcinoma cell lines. We report that both functional p53 and caspase-2 were required for the apoptotic response, which was preceded by translocation of nuclear caspase-2 to the cytoplasm. In the absence of functional p53, cisplatin treatment resulted in caspase-2-independent mitotic catastrophe followed by necrosis. In these cells, apoptotic functions could be restored by transient expression of wt p53. Hence, p53 appeared to act as a switch between apoptosis and mitotic catastrophe followed by necrosis-like lysis in this experimental model. Further, we show that inhibition of Chk2, and/or 14-3-3sigma deficiency, sensitized cells to undergo mitotic catastrophe upon treatment with DNA-damaging agents. However, apoptotic cell death seemed to be the final outcome of this process. Thus, we hypothesize that the final mode of cell death triggered by DNA damage in ovarian carcinoma cells is determined by the profile of proteins involved in the regulation of the cell cycle, such as p53- and Chk2-related proteins.     

A DNA vaccine candidate for <Emphasis Type="Italic">B. anthracis</Emphasis> immunization,pcDNA3.1+PA plasmid,induce Th1/Th2 mixed responses and protection in mice

The protective antigen (PA) of Bacillus anthracis (B. anthracis) is a potent immunogen and a candidate subunit vaccine. To address the question whether antibodies raised against PA following injection of pcDNA3.1+PA plasmid, encoding PA, can protect against virulent B. anthracis two different regimens of PA based vaccines (DNA and live spore) were used. The groups of BALB/c mice that received live spores of the Sterne strain, naked pcDNA3.1 and naked pcDNA3.1+PA were compared to control groups. All groups were injected three times with 30-day intervals. Two weeks after the last immunization, all mice were subjected to challenge with a pathogenic strain of B. anthracis (C2). Blood samples were taken before each injection and challenge. Evaluation of the sera by ELISA method showed that DNA immunization using pcDNA3.1+PA plasmid resulted in an antibody profile representative of a mixed Th1 and Th2 response, with a skewing to a Th1 response. The group which received the naked pcDNA3.1+PA had a survival rate of >80%. This challenge assay revealed that antibodies raised following DNA vaccination against PA can confer strong protection, and resistance against virulent species of B. anthracis.     

Detection and characterization of an ABC transporter in <Emphasis Type="Italic">Clostridium hathewayi</Emphasis>

An ABC transporter gene from Clostridium hathewayi is characterized. It has duplicated ATPase domains in addition to a transmembrane protein. Its deduced amino acid sequence has conserved functional domains with ATPase components of the multidrug efflux pump genes of several bacteria. Cloning this transporter gene into C. perfringens and E. coli resulted in decreased sensitivities of these bacteria to fluoroquinolones. It also decreased the accumulation and increased the efflux of ethidium bromide from cells containing the cloned gene. Carbonyl cyanide-m-chlorophenylhydrazone (CCCP) inhibited both accumulation and efflux of ethidium bromide from these cells. The ATPase mRNA was overexpressed in the fluoroquinolone-resistant strain when exposed to ciprofloxacin. This is the first report of an ABC transporter in C. hathewayi. An erratum to this article can be found at     

Spatial and technological variability in the carbon footprint of durum wheat production in Iran

Purpose

The purpose of this study was to quantify the spatial and technological variability in life cycle greenhouse gas (GHG) emissions, also called the carbon footprint, of durum wheat production in Iran.

Methods

The calculations were based on information gathered from 90 farms, each with an area ranging from 1 to 150 ha (average 16 ha). The carbon footprint of durum wheat was calculated by quantifying the biogenic GHG emissions of carbon loss from soil and biomass, as well as the GHG emissions from fertilizer application and machinery use, irrigation, transportation, and production of inputs (e.g., fertilizers, seeds, and pesticides). We used Spearman’s rank correlation to quantify the relative influence of technological variability (in crop yields, fossil GHG emissions, and N₂O emissions from fertilizer application) and spatial variability (in biogenic GHG emissions) on the variation of the carbon footprint of durum wheat.

Results and discussion

The average carbon footprint of 1 kg of durum wheat produced was 1.6 kg CO₂-equivalents with a minimum of 0.8 kg and a maximum of 3.0 kg CO₂-equivalents. The correlation analysis showed that variation in crop yield and fertilizer application, representing technological variability, accounted for the majority of the variation in the carbon footprint, respectively 76 and 21%. Spatial variation in biogenic GHG emissions, mainly resulting from differences in natural soil carbon stocks, accounted for 3% of the variation in the carbon footprint. We also observed a non-linear relationship between the carbon footprint and the yield of durum wheat that featured a scaling factor of ?2/3. This indicates that the carbon footprint of durum wheat production (in kg CO₂-eq kg^?1) typically decreases by 67% with a 100% increase in yield (in kg ha^?1 year^?1).

Conclusions

Various sources of variability, including variation between locations and technologies, can influence the results of life cycle assessments. We demonstrated that technological variability exerts a relatively large influence on the carbon footprint of durum wheat produced in Iran with respect to spatial variability. To increase the durum wheat yield at farms with relatively large carbon footprints, technologies such as site-specific nutrient application, combined tillage, and mechanized irrigation techniques should be promoted.

     

The critically endangered Asiatic cheetah <Emphasis Type="Italic">Acinonyx jubatus venaticus</Emphasis> in Iran: a review of recent distribution,and conservation status

Considerable effort has been put into conservation of the critically endangered Asiatic cheetah Acinonyx jubatus venaticus in Iran during the past few decades, and a thorough review of the species’ status, demography, range and conservation is provided here. We collated a large dataset of all verified occurrence data, photographic records and mortality cases since 1980 throughout the species’ range in Iran. Currently, the cheetah is distributed throughout the arid landscapes of the eastern half of Iran, but the limits of its current and past range as well as population trends are uncertain. Surveys of nearly 40 different areas resulted in 18 localities with confirmed presence of cheetahs in recent years. Camera trapping has been an effective tool to provide evidence of presence and status of cheetahs, revealing the species’ extremely low density and long inter-reserve movements. Together with photographic records, a total of 82 different cheetahs were detected during the 2000s in Iran. Protection status in most areas has been elevated by the Iran government. Asiatic cheetahs are highly vulnerable to extinction, mainly due to causalities mediated by herder persecution, poaching and road collisions as well as prey and habitat loss. Some efforts have been made to address these threats, but range expansion in recent years is a result of greater survey effort, rather than population recovery. We suggest that, despite conservation investment of the last 15 years, the species remains critically endangered on the verge of extinction.     

Thermodynamic Parameters and Influence of Kinetic Factors on the Self-Assembly of Acid-Soluble Collagen Nanofibrils

In this study, the acid-soluble collagen (ASC), extracted from the fish scales of the Caspian white fish (Rutilus Firisikutum) was studied. The thermo-gravimetric analysis (TGA) showed the maximum demineralization accomplished after 48 h of EDTA treatment. SDS-PAGE and FT-IR spectroscopy confirmed that extracted ASC was mainly type I collagen. FE-SEM images confirmed the porous and filamentary structure. The denaturation temperature (T_d) of ASC was 19 °C, and the transition heat achieved 9.6 J/g. Collagen self-assembly exhibit important potential because for biomedical applications and green technologies. Various inter- and intra-molecular no-covalent interactions such as hydrogen bonding, hydrophobic, electrostatic and Van der Waals interactions influence the formation of self-assembled collagen. Therefore, critical factors as concentration of ASC, temperature, pH, and ionic strength play crucial role in function integration and structural modulation. The impacts of those external triggers on the kinetic self-assembly of ASC demonstrated a two-phase kinetic process, a sigmoidal plot. ACS showed pronounced self-assembly behavior when temperature and concentration reach above 14 °C and 0.125 mg/ml, higher concentration and/or temperature could stimulate the ASC self-assembly. The optimum pH value for ASC self-assembly was pH = 7. The effect of ionic strength on ASC self-assembly showed the turbidity increases significantly in 131.2 mM salt concentration. The process of self-assembly is mainly driven by thermodynamics. The thermodynamic study of collagen self-assembly illustrated that the activation energy, E_a = 44.3 kJ/mol, the frequency factor, A = 117 × 10⁵ s^?1, the enthalpy transition, ΔH^? = 42.98 kJ/mol, and the entropy transition, ΔS^? = ?0.12 kJ/mol.K, respectively. These findings show that kinetics factors not only influence the self-assembly structure of ASC but also regulate the activation complex structure in the transition state.