Differences in the glycosylation of recombinant proteins expressed in HEK and CHO cells

Glycosylation is one of the most common posttranslational modifications of proteins. It has important roles for protein structure, stability and functions. In vivo the glycostructures influence pharmacokinetics and immunogenecity. It is well known that significant differences in glycosylation and glycostructures exist between recombinant proteins expressed in mammalian, yeast and insect cells. However, differences in protein glycosylation between different mammalian cell lines are much less well known. In order to examine differences in glycosylation in mammalian cells we have expressed 12 proteins in the two commonly used cell lines HEK and CHO. The cells were transiently transfected, and the expressed proteins were purified. To identify differences in glycosylation the proteins were analyzed on SDS-PAGE, isoelectric focusing (IEF), mass spectrometry and released glycans on capillary gel electrophoresis (CGE-LIF). For all proteins significant differences in the glycosylation were detected. The proteins migrated differently on SDS-PAGE, had different isoform patterns on IEF, showed different mass peak distributions on mass spectrometry and showed differences in the glycostructures detected in CGE. In order to verify that differences detected were attributed to glycosylation the proteins were treated with deglycosylating enzymes. Although, culture conditions induced minor changes in the glycosylation the major differences were between the two cell lines.     

Screening for Vulnerability in Older Cancer Patients: The ONCODAGE Prospective Multicenter Cohort Study

Background

Geriatric Assessment is an appropriate method for identifying older cancer patients at risk of life-threatening events during therapy. Yet, it is underused in practice, mainly because it is time- and resource-consuming. This study aims to identify the best screening tool to identify older cancer patients requiring geriatric assessment by comparing the performance of two short assessment tools the G8 and the Vulnerable Elders Survey (VES-13).

Patients and Methods

The diagnostic accuracy of the G8 and the (VES-13) were evaluated in a prospective cohort study of 1674 cancer patients accrued before treatment in 23 health care facilities. 1435 were eligible and evaluable. Outcome measures were multidimensional geriatric assessment (MGA), sensitivity (primary), specificity, negative and positive predictive values and likelihood ratios of the G8 and VES-13, and predictive factors of 1-year survival rate.

Results

Patient median age was 78.2 years (70-98) with a majority of females (69.8%), various types of cancer including 53.9% breast, and 75.8% Performance Status 0-1. Impaired MGA, G8, and VES-13 were 80.2%, 68.4%, and 60.2%, respectively. Mean time to complete G8 or VES-13 was about five minutes. Reproducibility of the two questionnaires was good. G8 appeared more sensitive (76.5% versus 68.7%, P =  0.0046) whereas VES-13 was more specific (74.3% versus 64.4%, P<0.0001). Abnormal G8 score (HR = 2.72), advanced stage (HR = 3.30), male sex (HR = 2.69) and poor Performance Status (HR = 3.28) were independent prognostic factors of 1-year survival.

Conclusion

With good sensitivity and independent prognostic value on 1-year survival, the G8 questionnaire is currently one of the best screening tools available to identify older cancer patients requiring geriatric assessment, and we believe it should be implemented broadly in daily practice. Continuous research efforts should be pursued to refine the selection process of older cancer patients before potentially life-threatening therapy.     

Microbiome mediating methane and nitrogen transformations in a subterranean estuary

Subterranean estuaries (STEs) are important coastal biogeochemical reactors facilitating unique niches for microbial communities. A common approach in determining STE greenhouse gas and nutrient fluxes is to use terrestrial endmembers, not accounting for microbially mediated transformations throughout the STE. As such, the microbial ecology and spatial distribution of specialists that cycle compounds in STEs remain largely underexplored. In this study, we applied 16S rRNA amplicon sequencing with paired biogeochemical characterisations to spatially evaluate microbial communities transforming greenhouse gases and nutrients in an STE. We show that methanogens are most prevalent at the terrestrial end (up to 2.81% relative abundance) concomitant to the highest porewater methane, carbon dioxide and dissolved organic carbon concentrations (0.41 ± 0.02 μM, 273.31 ± 6.05 μM and 0.51 ± 0.02 mM, respectively). Lower ammonium concentrations corresponded with abundant nitrifying and ammonia-oxidising prokaryotes in the mixing zone (up to 11.65% relative abundance). Methane, ammonium and dissolved organic carbon concentrations all decreased by >50% from the terrestrial to the oceanic end of the 15 m transect. This study highlights the STE's hidden microbiome zonation, as well as the importance of accounting for microbial transformations mitigating nutrient and greenhouse gas fluxes to the coastal ecosystems.     

Constitutive overexpression of the OsNAS gene family reveals single-gene strategies for effective iron- and zinc-biofortification of rice endosperm

Background

Rice is the primary source of food for billions of people in developing countries, yet the commonly consumed polished grain contains insufficient levels of the key micronutrients iron (Fe), zinc (Zn) and Vitamin A to meet daily dietary requirements. Experts estimate that a rice-based diet should contain 14.5 µg g⁻¹ Fe in endosperm, the main constituent of polished grain, but breeding programs have failed to achieve even half of that value. Transgenic efforts to increase the Fe concentration of rice endosperm include expression of ferritin genes, nicotianamine synthase genes (NAS) or ferritin in conjunction with NAS genes, with results ranging from two-fold increases via single-gene approaches to six-fold increases via multi-gene approaches, yet no approach has reported 14.5 µg g⁻¹ Fe in endosperm.

Methodology/Principal Findings

Three populations of rice were generated to constitutively overexpress OsNAS1, OsNAS2 or OsNAS3, respectively. Nicotianamine, Fe and Zn concentrations were significantly increased in unpolished grain of all three of the overexpression populations, relative to controls, with the highest concentrations in the OsNAS2 and OsNAS3 overexpression populations. Selected lines from each population had at least 10 µg g⁻¹ Fe in polished grain and two OsNAS2 overexpression lines had 14 and 19 µg g⁻¹ Fe in polished grain, representing up to four-fold increases in Fe concentration. Two-fold increases of Zn concentration were also observed in the OsNAS2 population. Synchrotron X-ray fluorescence spectroscopy demonstrated that OsNAS2 overexpression leads to significant enrichment of Fe and Zn in phosphorus-free regions of rice endosperm.

Conclusions

The OsNAS genes, particularly OsNAS2, show enormous potential for Fe and Zn biofortification of rice endosperm. The results demonstrate that rice cultivars overexpressing single rice OsNAS genes could provide a sustainable and genetically simple solution to Fe and Zn deficiency disorders affecting billions of people throughout the world.     

Trabecular architecture in the humeral metaphyses of non-avian reptiles (Crocodylia,Squamata and Testudines): Lifestyle,allometry and phylogeny

The lifestyle of extinct tetrapods is often difficult to assess when clear morphological adaptations such as swimming paddles are absent. According to the hypothesis of bone functional adaptation, the architecture of trabecular bone adapts sensitively to physiological loadings. Previous studies have already shown a clear relation between trabecular architecture and locomotor behavior, mainly in mammals and birds. However, a link between trabecular architecture and lifestyle has rarely been examined. Here, we analyzed trabecular architecture of different clades of reptiles characterized by a wide range of lifestyles (aquatic, amphibious, generalist terrestrial, fossorial, and climbing). Humeri of squamates, turtles, and crocodylians have been scanned with microcomputed tomography. We selected spherical volumes of interest centered in the proximal metaphyses and measured trabecular spacing, thickness and number, degree of anisotropy, average branch length, bone volume fraction, bone surface density, and connectivity density. Only bone volume fraction showed a significant phylogenetic signal and its significant difference between squamates and other reptiles could be linked to their physiologies. We found negative allometric relationships for trabecular thickness and spacing, positive allometries for connectivity density and trabecular number and no dependence with size for degree of anisotropy and bone volume fraction. The different lifestyles are well separated in the morphological space using linear discriminant analyses, but a cross-validation procedure indicated a limited predictive ability of the model. The trabecular bone anisotropy has shown a gradient in turtles and in squamates: higher values in amphibious than terrestrial taxa. These allometric scalings, previously emphasized in mammals and birds, seem to be valid for all amniotes. Discriminant analysis has offered, to some extent, a distinction of lifestyles, which however remains difficult to strictly discriminate. Trabecular architecture seems to be a promising tool to infer lifestyle of extinct tetrapods, especially those involved in the terrestrialization.     

The effects of sleep deprivation and time of day on cognitive performance

The extent to which the diurnal fluctuations of different cognitive processes could be affected by sleep loss may be explored to predict performance decrements observed in the real world. Twenty healthy male subjects voluntarily took part in 8 test sessions at 06:00, 10:00, 14:00, and 18:00 h, following either a night with or without sleep in random order. Measurements included oral temperature, simple reaction time, sign cancelation, Go/NoGo, and the Purdue pegboard test. The results indicate that simple reaction time and motor coordination had morning–afternoon variations closely following the rhythms of temperature and vigilance. Inhibitory attention (Go/NoGo) presented no morning–afternoon variations. Sleep deprivation may affect the profiles of cognitive performance depending on the processes solicited. Sustained and inhibitory attention are particularly affected in the morning (after 24 and 28 waking hours), while a complex task (visuo-motor coordination) would be affected after 32 waking hours only.     

Auditory anatomy of beaked whales and other odontocetes: Potential for cochlear stimulation via a “vibroacoustic duct mechanism”

Computed tomography (CT) and microcomputed tomography (microCT) were used to examine the structures involved in cochlear stimulation in odontocetes and terrestrial mammals. Cranial CT examined the osseous attachment of the skull to the tympanoperiotic complex (TPC) and the path of the endocranial foramen of the vestibulocochlear nerve (EFVN), which was assumed to contain the perilymphatic duct. Additional CTs of TPC were taken postextraction to examine the gross morphology of this structure. MicroCT was used to examine the acoustic windows of the cochlea, including the round and oval windows and the apertures of the cochlear and vestibular aqueducts. Cranial CT scans demonstrated an osseous connection between the skull and TPC in beaked whales and Physeter macrocephalus. EFVN traveled through a greater length of cranial bone and communicated more closely with the periotic bone in beaked whales than in other species. Ziphius cavirostris was observed to have a reduced medial sulcus of the mallear ridge (MSMR) and tympanic plate and an enlarged aperture of the cochlear aqueduct, respectively. The potential significance of these findings, including the role of the perilymphatic duct as a novel route of cochlear stimulation referred to as the “vibroacoustic duct mechanism,” are discussed.     

Partial Opsin Sequences Suggest UV-Sensitive Vision is Widespread in Caudata

Ultraviolet (UV) vision exists in several animal groups. Intuitively, one would expect this trait to be favoured in species living in bright environments, where UV light is the most present. However, UV sensitivity, as deduced from sequences of UV photoreceptors and/or ocular media transmittance, is also present in nocturnal species, raising questions about the selective pressure maintaining this perceptual ability. Amphibians are among the most nocturnal vertebrates but their visual ecology remains poorly understood relative to other groups. Perhaps because many of these species breed in environments that filter out a large part of UV radiation, physiological and behavioural studies of UV sensitivity in this group are scarce. We investigated the extent of UV vision in Caudata, the order of amphibians with the most nocturnal habits. We could recover sequences of the UV sensitive SWS1 opsin in 40 out of 58 species, belonging to 6 families. In all of these species, the evidence suggests the presence of functional SWS1 opsins under purifying selection, potentially allowing UV vision. Interestingly, most species whose opsin genes failed to amplify exhibited particular ecological features that could drive the loss of UV vision. This likely wide distribution of functional UV photoreceptors in Caudata sheds a new light on the visual ecology of amphibians and questions the function of UV vision in nocturnal animal species.