Cell type-specific and site directed N-glycosylation pattern of FcγRIIIa

Human leukocyte receptor IIIa (hFcγRIIIa) plays a prominent role in the elimination of tumor cells by antibody-based cancer therapies. In previous studies, a major impact of the presence of carbohydrates at Asn-162 on the binding between the receptor and the Fc part of wild type fucosylated or glycoengineered nonfucosylated antibodies has been shown. In this study, we performed a site directed carbohydrate analysis at hFcγRIIIa derived from human embryonic kidney (HEK) and Chinese hamster ovary (CHO) cells, respectively. Using mass spectrometry (MS) and a multienzyme protein digest, we analyzed the proteolysis-generated glycopeptides in detail. We could show that hFcγRIIIa expressed by HEK cells was mostly bearing multifucosylated biantennary Asn162-glycans with a major fraction terminating with GalNAc residues replacing the more common Gal. We could demonstrate that the glycan antennae with terminal GalNAc could be sialylated as indicated by a novel reporter ion HexNAcHexNAcNeuAc(+) (m/z 698.28) using a source induced dissociation (SID) scan in the MS cycle. In contrast to the hFcγRIIIa Asn-162 glycosylation pattern from HEK cells, the CHO cells derived receptor contains bi- and triantennary galactosylated and highly sialylated carbohydrates. Our data suggest that the type of expression host system was a dominating factor for formation of distinct glycopatterns of hFcγRIIIa, while the protein sequence and the site of glycosylation remained unchanged for both types of cells. Using surface plasmon resonance (SPR) interaction analysis, we show that the cell type and site specific glycosylation pattern of hFcγRIIIa influences its binding behavior to immunoglobulin molecules.     

Digital transformation—life cycle assessment of digital services,multifunctional devices and cloud computing

The International Journal of Life Cycle Assessment -     

Recombinant adeno-associated virus type 2-mediated gene delivery into the <Emphasis Type="Italic">Rpe65</Emphasis><Superscript>-/-</Superscript> knockout mouse eye results in limited rescue

Background  

Leber's congenital amaurosis (LCA) is a severe form of retinal dystrophy. Mutations in the RPE65 gene, which is abundantly expressed in retinal pigment epithelial (RPE) cells, account for approximately 10–15% of LCA cases. In this study we used the high turnover, and rapid breeding and maturation time of the Rpe65 ^-/- knockout mice to assess the efficacy of using rAAV-mediated gene therapy to replace the disrupted RPE65 gene. The potential for rAAV-mediated gene treatment of LCA was then analyzed by determining the pattern of RPE65 expression, the physiological and histological effects that it produced, and any improvement in visual function.     

Pooled sample-based GWAS: a cost-effective alternative for identifying colorectal and prostate cancer risk variants in the Polish population

Background

Prostate cancer (PCa) and colorectal cancer (CRC) are the most commonly diagnosed cancers and cancer-related causes of death in Poland. To date, numerous single nucleotide polymorphisms (SNPs) associated with susceptibility to both cancer types have been identified, but their effect on disease risk may differ among populations.

Methods

To identify new SNPs associated with PCa and CRC in the Polish population, a genome-wide association study (GWAS) was performed using DNA sample pools on Affymetrix Genome-Wide Human SNP 6.0 arrays. A total of 135 PCa patients and 270 healthy men (PCa sub-study) and 525 patients with adenoma (AD), 630 patients with CRC and 690 controls (AD/CRC sub-study) were included in the analysis. Allele frequency distributions were compared with t-tests and χ²-tests. Only those significantly associated SNPs with a proxy SNP (p<0.001; distance of 100 kb; r²>0.7) were selected. GWAS marker selection was conducted using PLINK. The study was replicated using extended cohorts of patients and controls. The association with previously reported PCa and CRC susceptibility variants was also examined. Individual patients were genotyped using TaqMan SNP Genotyping Assays.

Results

The GWAS selected six and 24 new candidate SNPs associated with PCa and CRC susceptibility, respectively. In the replication study, 17 of these associations were confirmed as significant in additive model of inheritance. Seven of them remained significant after correction for multiple hypothesis testing. Additionally, 17 previously reported risk variants have been identified, five of which remained significant after correction.

Conclusion

Pooled-DNA GWAS enabled the identification of new susceptibility loci for CRC in the Polish population. Previously reported CRC and PCa predisposition variants were also identified, validating the global nature of their associations. Further independent replication studies are required to confirm significance of the newly uncovered candidate susceptibility loci.     

Changes in temperature sensitivity of spring phenology with recent climate warming in Switzerland are related to shifts of the preseason

The spring phenology of plants in temperate regions strongly responds to spring temperatures. Climate warming has caused substantial phenological advances in the past, but trends to be expected in the future are uncertain. A simple indicator is temperature sensitivity, the phenological advance statistically associated with a 1°C warmer mean temperature during the “preseason”, defined as the most temperature‐sensitive period preceding the phenological event. Recent analyses of phenological records have shown a decline in temperature sensitivity of leaf unfolding, but underlying mechanisms were not clear. Here, we propose that climate warming can reduce temperature sensitivity simply by reducing the length of the preseason due to faster bud development during this time period, unless the entire preseason shifts forward so that its temperature does not change. We derive these predictions theoretically from the widely used “thermal time model” for bud development and test them using data for 19 phenological events recorded in 1970–2012 at 108 stations spanning a 1600 m altitudinal range in Switzerland. We consider how temperature sensitivity, preseason start, preseason length and preseason temperature change (i) with altitude, (ii) between the periods 1970–1987 and 1995–2012, which differed mainly in spring temperatures, and (iii) between two non‐consecutive sets of 18 years that differed mainly in winter temperatures. On average, temperature sensitivity increased with altitude (colder climate) and was reduced in years with warmer springs, but not in years with warmer winters. These trends also varied among species. Decreasing temperature sensitivity in warmer springs was associated with a limited forward shift of preseason start, higher temperatures during the preseason and reduced preseason length, but not with reduced winter chilling. Our results imply that declining temperature sensitivity can result directly from spring warming and does not necessarily indicate altered physiological responses or stronger constraints such as reduced winter chilling.     

Trait differentiation and adaptation of plants along elevation gradients

Studies of genetic adaptation in plant populations along elevation gradients in mountains have a long history, but there has until now been neither a synthesis of how frequently plant populations exhibit adaptation to elevation nor an evaluation of how consistent underlying trait differences across species are. We reviewed studies of adaptation along elevation gradients (i) from a meta‐analysis of phenotypic differentiation of three traits (height, biomass and phenology) from plants growing in 70 common garden experiments; (ii) by testing elevation adaptation using three fitness proxies (survival, reproductive output and biomass) from 14 reciprocal transplant experiments; (iii) by qualitatively assessing information at the molecular level, from 10 genomewide surveys and candidate gene approaches. We found that plants originating from high elevations were generally shorter and produced less biomass, but phenology did not vary consistently. We found significant evidence for elevation adaptation in terms of survival and biomass, but not for reproductive output. Variation in phenotypic and fitness responses to elevation across species was not related to life history traits or to environmental conditions. Molecular studies, which have focussed mainly on loci related to plant physiology and phenology, also provide evidence for adaptation along elevation gradients. Together, these studies indicate that genetically based trait differentiation and adaptation to elevation are widespread in plants. We conclude that a better understanding of the mechanisms underlying adaptation, not only to elevation but also to environmental change, will require more studies combining the ecological and molecular approaches.     

Teaching life cycle assessment in higher education

The International Journal of Life Cycle Assessment - Scientific Life Cycle Assessment (LCA) literature provides some examples of LCA teaching in higher education, but not a structured overview of...     

Climate change fingerprints in recent European plant phenology

A paper published in Global Change Biology in 2006 revealed that phenological responses in 1971–2000 matched the warming pattern in Europe, but a lack of chilling and adaptation in farming may have reversed these findings. Therefore, for 1951–2018 in a corresponding data set, we determined changes as linear trends and analysed their variation by plant traits/groups, across season and time as well as their attribution to warming following IPCC methodology. Although spring and summer phases in wild plants advanced less (maximum advances in 1978–2007), more (~90%) and more significant (~60%) negative trends were present, being stronger in early spring, at higher elevations, but smaller for nonwoody insect‐pollinated species. These trends were strongly attributable to winter and spring warming. Findings for crop spring phases were similar, but were less pronounced. There were clearer and attributable signs for a delayed senescence in response to winter and spring warming. These changes resulted in a longer growing season, but a constant generative period in wild plants and a shortened one in agricultural crops. Phenology determined by farmers’ decisions differed noticeably from the purely climatic driven phases with smaller percentages of advancing (~75%) trends, but farmers’ spring activities were the only group with reinforced advancement, suggesting adaptation. Trends in farmers’ spring and summer activities were very likely/likely associated with the warming pattern. In contrast, the advance in autumn farming phases was significantly associated with below average summer warming. Thus, under ongoing climate change with decreased chilling the advancing phenology in spring and summer is still attributable to warming; even the farmers’ activities in these seasons mirror, to a lesser extent, the warming. Our findings point to adaptation to climate change in agriculture and reveal diverse implications for terrestrial ecosystems; the strong attribution supports the necessary mediation of warming impacts to the general public.     

Yos9 protein is essential for degradation of misfolded glycoproteins and may function as lectin in ERAD

The Htm1/EDEM protein has been proposed to act as a "degradation lectin" for endoplasmic reticulum-associated degradation (ERAD) of misfolded glycoproteins. In this study, we provide genetic and biochemical evidence that Yos9 protein in Saccharomyces cerevisiae is essential for efficient degradation of mutant glycoproteins. Yos9 is a member of the OS-9 protein family, which is conserved among eukaryotes and shows similarities with mannose-6-phosphate receptors (MPRs). We found that amino acids conserved among OS-9 family members and MPRs were essential for Yos9 protein function. Immunoprecipitation showed that Yos9 specifically associated with misfolded carboxypeptidase Y (CPY*), an ERAD substrate, but only when it carried Man8GlcNAc2 or Man5GlcNAc2 N-glycans. Our experiments further suggested that Yos9 acts in the same pathway as Htm1/EDEM. Yos9 protein is important for glycoprotein degradation and may act via its MRH domain as a degradation lectin-like protein in the glycoprotein degradation pathway.     

Short-patch correction of C/C mismatches in human cells

We examined whether the human nucleotide excision repair complex, which is specialized on the removal of bulky DNA adducts, also displays a correcting activity on base mismatches. The cytosine/cytosine (C/C) lesion was used as a model substrate to monitor the correction of base mismatches in human cells. Fibroblasts with different repair capabilities were transfected with shuttle vectors that contain a site-directed C/C mismatch in the replication origin, accompanied by an additional C/C mismatch in one of the flanking sequences that are not essential for replication. Analysis of the vector progeny obtained from these doubly modified substrates revealed that C/C mismatches were eliminated before DNA synthesis not only in the repair-proficient background, but also when the target cells carried a genetic defect in long-patch mismatch repair, in nucleotide excision repair, or when both pathways were deleted. Furthermore, cells deficient for long-patch mismatch repair as well as a cell line that combines mismatch and nucleotide excision repair defects were able to correct multiple C/C mispairs, placed at distances of 21–44 nt, in an independent manner, such that the removal of each lesion led to individual repair patches. These results support the existence of a concurrent short-patch mechanism that rectifies C/C mismatches.