Embryonic stem-cell culture as a tool for developmental cell biology

The cell biology of the early processes of mammalian embryogenesis, such as germ-layer formation, has been technically challenging to study owing to the size and accessibility of mammalian embryos. Embryonic stem cells, which can generate the three germ layers in vitro, are useful for studying embryogenesis at the cellular level. So, how can the study of embryonic stem cells and their differentiation provide a deeper understanding of the cell biology of early development?     

Endocytosis: clathrin-mediated membrane budding

Clathrin-dependent endocytosis is the major pathway for the uptake of nutrients and signaling molecules in higher eukaryotic cells. The long-held tenet that clathrin-coated vesicles are created from flat coated plasma membrane patches by a sequential process of invagination, bud formation and fission recently received strong support from the results of advanced live cell fluorescence microscopy. The data on the critical components that deform the plasma membrane locally into a coated bud suggest that membrane bending is a team effort requiring membrane-curving protein domains, actin dynamics and, last but not least, clathrin. The scission step requires the mechano-enzymatic function of dynamin, actin dynamics and possibly myosin motor proteins. Finally, a burst of auxilin/GAK initiates the uncoating of the vesicle.     

Sensitive ELISA detection of amyloid-beta protofibrils in biological samples

Amyloid-beta (Abeta) protofibrils are known intermediates of the in vitro Abeta aggregation process and the protofibrillogenic Arctic mutation (APPE693G) provides clinical support for a pathogenic role of Abeta protofibrils in Alzheimer's disease (AD). To verify their in vivo relevance and to establish a quantitative Abeta protofibril immunoassay, Abeta conformation dependent monoclonal antibodies were generated. One of these antibodies, mAb158 (IgG2a), was used in a sandwich ELISA to specifically detect picomolar concentrations of Abeta protofibrils without interference from Abeta monomers or the amyloid precursor protein (APP). The specificity and biological significance of this ELISA was demonstrated using cell cultures and transgenic mouse models expressing human APP containing the Swedish mutation (APPKN670/671ML), or the Swedish and Arctic mutation in combination. The mAb158 sandwich ELISA analysis revealed presence of Abeta protofibrils in both cell and animal models, proving that Abeta protofibrils are formed not only in vitro, but also in vivo. Furthermore, elevated Abeta protofibril levels in the Arctic-Swedish samples emphasize the usefulness of the Arctic mutation as a model of enhanced protofibril formation. This assay provides a novel tool for investigating the role of Abeta protofibrils in AD and has the potential of becoming an important diagnostic assay.     

Aquaporin-1 channel function is positively regulated by protein kinase C

Aquaporin-1 (AQP1) channels contribute to osmotically induced water transport in several organs including the kidney and serosal membranes such as the peritoneum and the pleura. In addition, AQP1 channels have been shown to conduct cationic currents upon stimulation by cyclic nucleotides. To date, the short term regulation of AQP1 function by other major intracellular signaling pathways has not been studied. In the present study, we therefore investigated the regulation of AQP1 by protein kinase C. AQP1 wild type channels were expressed in Xenopus oocytes. Water permeability was assessed by hypotonic challenges. Activation of protein kinase C (PKC) by 1-oleoyl-2-acetyl-sn-glycerol (OAG) induced a marked increase of AQP1-dependent water permeability. This regulation was abolished in mutated AQP1 channels lacking both consensus PKC phosphorylation sites Thr(157) and Thr(239) (termed AQP1 DeltaPKC). AQP1 cationic currents measured with double-electrode voltage clamp were markedly increased after pharmacological activation of PKC by either OAG or phorbol 12-myristate 13-acetate. Deletion of either Thr(157) or Thr(239) caused a marked attenuation of PKC-dependent current increases, and deletion of both phosphorylation sites in AQP1 DeltaPKC channels abolished the effect. In vitro phosphorylation studies with synthesized peptides corresponding to amino acids 154-168 and 236-250 revealed that both Thr(157) and Thr(239) are phosphorylated by PKC. Upon stimulation by cyclic nucleotides, AQP1 wild type currents exhibited a strong activation. This regulation was not affected after deletion of PKC phosphorylation sites in AQP1 DeltaPKC channels. In conclusion, this is the first study to show that PKC positively regulates both water permeability and ionic conductance of AQP1 channels. This new pathway of AQP1 regulation is independent of the previously described cyclic nucleotide pathway and may contribute to the PKC stimulation of AQP1-modulated processes such as endothelial permeability, angiogenesis, and urine concentration.     

Organization of the pronephric kidney revealed by large-scale gene expression mapping

Background

The pronephros, the simplest form of a vertebrate excretory organ, has recently become an important model of vertebrate kidney organogenesis. Here, we elucidated the nephron organization of the Xenopus pronephros and determined the similarities in segmentation with the metanephros, the adult kidney of mammals.

Results

We performed large-scale gene expression mapping of terminal differentiation markers to identify gene expression patterns that define distinct domains of the pronephric kidney. We analyzed the expression of over 240 genes, which included members of the solute carrier, claudin, and aquaporin gene families, as well as selected ion channels. The obtained expression patterns were deposited in the searchable European Renal Genome Project Xenopus Gene Expression Database. We found that 112 genes exhibited highly regionalized expression patterns that were adequate to define the segmental organization of the pronephric nephron. Eight functionally distinct domains were discovered that shared significant analogies in gene expression with the mammalian metanephric nephron. We therefore propose a new nomenclature, which is in line with the mammalian one. The Xenopus pronephric nephron is composed of four basic domains: proximal tubule, intermediate tubule, distal tubule, and connecting tubule. Each tubule may be further subdivided into distinct segments. Finally, we also provide compelling evidence that the expression of key genes underlying inherited renal diseases in humans has been evolutionarily conserved down to the level of the pronephric kidney.

Conclusion

The present study validates the Xenopus pronephros as a genuine model that may be used to elucidate the molecular basis of nephron segmentation and human renal disease.     

Hydration, thermoregulation, and performance effects of two sport drinks during soccer training sessions

In the present study, we aimed to compare the thermoregulatory response and soccer-specific training performance aspects of two commercially available sport drinks, both of similar carbohydrate concentration, but one containing 5.2% glycerol. Ten players participated in two similar outdoor training sessions and were randomly assigned to each of two drinks: a carbohydrate (C) beverage or a carbohydrate-glycerol (CG) beverage. Players consumed 500 mL of C or CG 30 minutes pre-exercise and at half-time. Pre- and postexercise body mass, core temperature (CT), and heart rate (HR) were recorded, and urine and blood samples were taken. No difference was observed between days for wet bulb globe temperature (session 1: 17.0 +/- 1.1 degrees C, session 2: 16.9 +/- 1.1 degrees C; P = 0.944). The degree of dehydration (% Delta BM) was greater after the C trial (P = 0.041). Similarly, percent change in plasma volume was greater in the C trial (P = 0.049). No overall main affect was observed between CT and mean exercise HRs during either training session (CT: P = 0.350; mean HR: P = 0.256), and there was no difference observed between groups in time to failure during the session-ending fatigue test (P = 0.547). Ingestion of a CG beverage provided players with better hydration than C alone. However, if training sessions are short (<75 minute), with adequate time for recovery, both drinks are sufficient for maintaining performance intensities during soccer-specific training.     

Localization of Microfibrillar-Associated Protein 4 (MFAP4) in Human Tissues: Clinical Evaluation of Serum MFAP4 and Its Association with Various Cardiovascular Conditions

Microfibrillar-associated protein 4 (MFAP4) is located in the extracellular matrix (ECM). We sought to identify tissues with high levels of MFAP4 mRNA and MFAP4 protein expression. Moreover, we aimed to evaluate the significance of MFAP4 as a marker of cardiovascular disease (CVD) and to correlate MFAP4 with other known ECM markers, such as fibulin-1, osteoprotegerin (OPG), and osteopontin (OPN). Quantitative real-time PCR demonstrated that MFAP4 mRNA was more highly expressed in the heart, lung, and intestine than in other elastic tissues. Immunohistochemical studies demonstrated high levels of MFAP4 protein mainly at sites rich in elastic fibers and within blood vessels in all tissues investigated. The AlphaLISA technique was used to determine serum MFAP4 levels in a clinical cohort of 172 patients consisting of 5 matched groups with varying degrees of CVD: 1: patients with ST elevation myocardial infarction (STEMI), 2: patients with non-STEMI, 3: patients destined for vascular surgery because of various atherosclerotic diseases (stable atherosclerotic disease), 4: apparently healthy individuals with documented coronary artery calcification (CAC-positive), and 5: apparently healthy individuals without signs of coronary artery calcification (CAC-negative). Serum MFAP4 levels were significantly lower in patients with stable atherosclerotic disease than CAC-negative individuals (p<0.05). Furthermore, lower serum MFAP4 levels were present in patients with stable atherosclerotic disease compared with STEMI and non-STEMI patients (p<0.05). In patients with stable atherosclerotic disease, positive correlations between MFAP4 and both fibulin-1 (ρ = 0.50; p = 0.0244) and OPG (ρ = 0.62; p = 0.0014) were found. Together, these results indicate that MFAP4 is mainly located in elastic fibers and is highly expressed in blood vessels. The present study suggests that serum MFAP4 varies in groups of patients with different cardiovascular conditions. Further studies are warranted to describe the role of serum MFAP4 as a biomarker of stable atherosclerotic disease.     

Quantifying Missing Heritability at Known GWAS Loci

Recent work has shown that much of the missing heritability of complex traits can be resolved by estimates of heritability explained by all genotyped SNPs. However, it is currently unknown how much heritability is missing due to poor tagging or additional causal variants at known GWAS loci. Here, we use variance components to quantify the heritability explained by all SNPs at known GWAS loci in nine diseases from WTCCC1 and WTCCC2. After accounting for expectation, we observed all SNPs at known GWAS loci to explain more heritability than GWAS-associated SNPs on average (). For some diseases, this increase was individually significant: for Multiple Sclerosis (MS) () and for Crohn''s Disease (CD) (); all analyses of autoimmune diseases excluded the well-studied MHC region. Additionally, we found that GWAS loci from other related traits also explained significant heritability. The union of all autoimmune disease loci explained more MS heritability than known MS SNPs () and more CD heritability than known CD SNPs (), with an analogous increase for all autoimmune diseases analyzed. We also observed significant increases in an analysis of Rheumatoid Arthritis (RA) samples typed on ImmunoChip, with more heritability from all SNPs at GWAS loci () and more heritability from all autoimmune disease loci () compared to known RA SNPs (including those identified in this cohort). Our methods adjust for LD between SNPs, which can bias standard estimates of heritability from SNPs even if all causal variants are typed. By comparing adjusted estimates, we hypothesize that the genome-wide distribution of causal variants is enriched for low-frequency alleles, but that causal variants at known GWAS loci are skewed towards common alleles. These findings have important ramifications for fine-mapping study design and our understanding of complex disease architecture.     

Structure and Function of Bacterial Communities Emerging from Different Sources under Identical Conditions

The aim of this study was to compare two major hypotheses concerning the formation of bacterial community composition (BCC) at the local scale, i.e., whether BCC is determined by the prevailing local environmental conditions or by “metacommunity processes.” A batch culture experiment where bacteria from eight distinctly different aquatic habitats were regrown under identical conditions was performed to test to what extent similar communities develop under similar selective pressure. Differently composed communities emerged from different inoculum communities, as determined by terminal restriction fragment length polymorphism analysis of the 16S rRNA gene. There was no indication that similarity increased between communities upon growth under identical conditions compared to that for growth at the ambient sampling sites. This suggests that the history and distribution of taxa within the source communities were stronger regulating factors of BCC than the environmental conditions. Moreover, differently composed communities were different with regard to specific functions, such as enzyme activities, but maintained similar broad-scale functions, such as biomass production and respiration.     

A test of the "sexy son" hypothesis: sons of polygynous collared flycatchers do not inherit their fathers' mating status

According to the original "sexy son" hypothesis, a female may benefit from pairing with an already-mated male despite a reduction in fecundity because her sons inherit their father's attractiveness. We used data from a long-term study of collared flycatchers (Ficedula albicollis) collected during 24 years to test this prediction. Our results show that the sons of polygynously mated females fledged in poor condition and therefore did not inherit their father's large forehead patch (a condition-dependent display trait) or mating status. From the female's perspective, polygynous pairing resulted in fewer recruited grandchildren than did a monogamous pairing. The reproductive value of sons did not outweigh the fecundity costs of polygyny because the low paternal care reduced the attractiveness of sons. When there are long-lasting parental effects on offspring attractiveness, costs of polygyny may include the production of nonsexy sons.