In Vivo Synthesis of Mammalian-Like, Hybrid-Type N-Glycans in Pichia pastoris

The Pichia pastoris N-glycosylation pathway is only partially homologous to the pathway in human cells. In the Golgi apparatus, human cells synthesize complex oligosaccharides, whereas Pichia cells form mannose structures that can contain up to 40 mannose residues. This hypermannosylation of secreted glycoproteins hampers the downstream processing of heterologously expressed glycoproteins and leads to the production of protein-based therapeutic agents that are rapidly cleared from the blood because of the presence of terminal mannose residues. Here, we describe engineering of the P. pastoris N-glycosylation pathway to produce nonhyperglycosylated hybrid glycans. This was accomplished by inactivation of OCH1 and overexpression of an α-1,2-mannosidase retained in the endoplasmic reticulum and N-acetylglucosaminyltransferase I and β-1,4-galactosyltransferase retained in the Golgi apparatus. The engineered strain synthesized a nonsialylated hybrid-type N-linked oligosaccharide structure on its glycoproteins. The procedures which we developed allow glycan engineering of any P. pastoris expression strain and can yield up to 90% homogeneous protein-linked oligosaccharides.     

Trigger factor interacts with the signal peptide of nascent Tat substrates but does not play a critical role in Tat-mediated export.

Twin-arginine translocation (Tat)-mediated protein transport across the bacterial cytoplasmic membrane occurs only after synthesis and folding of the substrate protein that contains a signal peptide with a characteristic twin-arginine motif. This implies that premature contact between the Tat signal peptide and the Tat translocon in the membrane must be prevented. We used site-specific photo-crosslinking to demonstrate that the signal peptide of nascent Tat proteins is in close proximity to the chaperone and peptidyl-prolyl isomerase trigger factor (TF). The contact with TF was strictly dependent on the context of the translating ribosome, started early in biogenesis when the nascent chain left the ribosome near L23, and persisted until the chain reached its full length. Despite this exclusive and prolonged contact, depletion or overexpression of TF had little effect on the kinetics and efficiency of the Tat export process.     

Heritability of metabolic syndrome traits in a large population-based sample

Heritability estimates of metabolic syndrome traits vary widely across studies. Some studies have suggested that the contribution of genes may vary with age or sex. We estimated the heritability of 11 metabolic syndrome-related traits and height as a function of age and sex in a large population-based sample of twin families (N = 2,792–27,021, for different traits). A moderate-to-high heritability was found for all traits [from H² = 0.47 (insulin) to H² = 0.78 (BMI)]. The broad-sense heritability (H²) showed little variation between age groups in women; it differed somewhat more in men (e.g., for glucose, H² = 0.61 in young females, H² = 0.56 in older females, H² = 0.64 in young males, and H²= 0.27 in older males). While nonadditive genetic effects explained little variation in the younger subjects, nonadditive genetic effects became more important at a greater age. Our findings show that in an unselected sample (age range, ∼18–98 years), the genetic contribution to individual differences in metabolic syndrome traits is moderate to large in both sexes and across age. Although the prevalence of the metabolic syndrome has greatly increased in the past decades due to lifestyle changes, our study indicates that most of the variation in metabolic syndrome traits between individuals is due to genetic differences.     

The Probability to Initiate X Chromosome Inactivation Is Determined by the X to Autosomal Ratio and X Chromosome Specific Allelic Properties

Background

In female mammalian cells, random X chromosome inactivation (XCI) equalizes the dosage of X-encoded gene products to that in male cells. XCI is a stochastic process, in which each X chromosome has a probability to be inactivated. To obtain more insight in the factors setting up this probability, we studied the role of the X to autosome (X∶A) ratio in initiation of XCI, and have used the experimental data in a computer simulation model to study the cellular population dynamics of XCI.

Methodology/Principal Findings

To obtain more insight in the role of the X∶A ratio in initiation of XCI, we generated triploid mouse ES cells by fusion of haploid round spermatids with diploid female and male ES cells. These fusion experiments resulted in only XXY triploid ES cells. XYY and XXX ES lines were absent, suggesting cell death related either to insufficient X-chromosomal gene dosage (XYY) or to inheritance of an epigenetically modified X chromosome (XXX). Analysis of active (Xa) and inactive (Xi) X chromosomes in the obtained triploid XXY lines indicated that the initiation frequency of XCI is low, resulting in a mixed population of XaXiY and XaXaY cells, in which the XaXiY cells have a small proliferative advantage. This result, and findings on XCI in diploid and tetraploid ES cell lines with different X∶A ratios, provides evidence that the X∶A ratio determines the probability for a given X chromosome to be inactivated. Furthermore, we found that the kinetics of the XCI process can be simulated using a probability for an X chromosome to be inactivated that is proportional to the X∶A ratio. These simulation studies re-emphasize our hypothesis that the probability is a function of the concentration of an X-encoded activator of XCI, and of X chromosome specific allelic properties determining the threshold for this activator.

Conclusions

The present findings reveal that the probability for an X chromosome to be inactivated is proportional to the X∶A ratio. This finding supports the presence of an X-encoded activator of the XCI process.     

Specificity of the mutualistic association between actinomycete bacteria and two sympatric species of Acromyrmex leaf-cutting ants

Acromyrmex leaf-cutting ants maintain two highly specialized, vertically transmitted mutualistic ectosymbionts: basidiomycete fungi that are cultivated for food in underground gardens and actinomycete Pseudonocardia bacteria that are reared on the cuticle to produce antibiotics that suppress the growth of Escovopsis parasites of the fungus garden. Mutualism stability has been hypothesized to benefit from genetic uniformity of symbionts, as multiple coexisting strains are expected to compete and, thus, reduce the benefit of the symbiosis. However, the Pseudonocardia symbionts are likely to be involved in Red-Queen-like antagonistic co-evolution with Escovopsis so that multiple strains per host might be favoured by selection provided the cost of competition between bacterial strains is low. We examined the genetic uniformity of the Pseudonocardia symbionts of two sympatric species of Acromyrmex ants by comparing partial sequences of the nuclear Elongation Factor-Tu gene. We find no genetic variation in Pseudonocardia symbionts among nest mate workers, neither in Acromyrmex octospinosus, where colonies are founded by a single queen, nor in Acromyrmex echinatior, where mixing of bacterial lineages might happen when unrelated queens cofound a colony. We further show that the two ant species maintain the same pool of Pseudonocardia symbionts, indicating that horizontal transmission occasionally occurs, and that this pool consists of two distinct clades of closely related Pseudonocardia strains. Our finding that individual colonies cultivate a single actinomycete strain is in agreement with predictions from evolutionary theory on host-symbiont conflict over symbiont mixing, but indicates that there may be constraints on the effectiveness of the bacterial symbionts on an evolutionary timescale.     

Joining the loops: beta-globin gene regulation

The mammalian beta-globin locus is a multigene locus containing several globin genes and a number of regulatory elements. During development, the expression of the genes changes in a process called "switching." The most important regulatory element in the locus is the locus control region (LCR) upstream of the globin genes that is essential for high-level expression of these genes. The discovery of the LCR initially raised the question how this element could exert its effect on the downstream globin genes. The question was solved by the finding that the LCR and activate globin genes are in physical contact, forming a chromatin structure named the active chromatin hub (ACH). Here we discuss the significance of ACH formation, provide an overview of the proteins implicated in chromatin looping at the beta-globin locus, and evaluate the relationship between nuclear organization and beta-globin gene expression.     

Adherence to Internet-Based and Face-to-Face Cognitive Behavioural Therapy for Depression: A Meta-Analysis

Background

Internet-based cognitive behavioural therapy (iCBT) is an effective and acceptable treatment for depression, especially when it includes guidance, but its treatment adherence has not yet been systematically studied. We conducted a meta-analysis, comparing the adherence to guided iCBT with the adherence to individual face-to-face CBT.

Methods

Studies were selected from a database of trials that investigate treatment for adult depression (see www.evidencebasedpsychotherapies.org), updated to January 2013. We identified 24 studies describing 26 treatment conditions (14 face-to-face CBT, 12 guided iCBT), by means of these inclusion criteria: targeting depressed adults, no comorbid somatic disorder or substance abuse, community recruitment, published in the year 2000 or later. The main outcome measure was the percentage of completed sessions. We also coded the percentage of treatment completers (separately coding for 100% or at least 80% of treatment completed).

Results

We did not find studies that compared guided iCBT and face-to-face CBT in a single trial that met our inclusion criteria. Face-to-face CBT treatments ranged from 12 to 28 sessions, guided iCBT interventions consisted of 5 to 9 sessions. Participants in face-to-face CBT completed on average 83.9% of their treatment, which did not differ significantly from participants in guided iCBT (80.8%, P  =  .59). The percentage of completers (total intervention) was significantly higher in face-to-face CBT (84.7%) than in guided iCBT (65.1%, P < .001), as was the percentage of completers of 80% or more of the intervention (face-to-face CBT: 85.2%, guided iCBT: 67.5%, P  =  .003). Non-completers of face-to-face CBT completed on average 24.5% of their treatment, while non-completers of guided iCBT completed on average 42.1% of their treatment.

Conclusion

We did not find studies that compared guided iCBT and face-to-face CBT in a single trial. Adherence to guided iCBT appears to be adequate and could be equal to adherence to face-to-face CBT.     

Immunocytochemical localization of epidermal growth factor receptor in early embryos of the Japanese medaka fish (Oryzias latipes)

This study was undertaken to localize epidermal growth factor receptor (EGFR) during early development of Japanese medaka embryos using immunocytochemistry. Specific staining was observed in all stages studied. All of the cells of the embryonic disc from the germinal disc (1 cell) through the late high blastula stages stained moderately for EGFR. Beginning with the flat blastula stage, the surface and lateral cells of the embryonic disc and the cells migrating around the yolk stained intensely for EGFR, and this continued throughout the study period. The presence of the keel at the late gastrula stage did not affect the moderate staining of the majority of the embryonic disc cells. When somites first appeared, the keel region stained less intensely than before, but scattered individual cells stained intensely for EGFR. Embryos with 12 somites had a neural tube that was lightly stained except for a few intensely stained individual cells. The neural tube, notochord and somites in 24-somite embryos lacked immunostaining. However, the surface epithelium, aorta, intestinal epithelium and pronephric duct demonstrated EGFR immunostaining. This study demonstrates that EGFR is present during medaka development and supports the hypothesis that EGFR ligands are important during cleavage, gastrulation and early organogenesis.