Reproductive state does not influence activity budgets of eusocial Ansell's mole-rats, Fukomys anselli (Rodentia, Bathyergidae): A study of locomotor activity by means of RFID

Ansell's mole-rats (Fukomys anselli) live in families of a breeding pair and several overlapping generations of their non-breeding offspring. Locomotor activity of 47 Ansell's mole-rats from six laboratory families was recorded for a week using radio frequency identification (RFID). This technique allowed monitoring each animal's activity without isolating them from other members of their family. Animals were active about 17% of the recording time, and older animals were significantly less active than younger ones. Females tended to be slightly more active than males. There was no significant difference in the amount of activity between breeding and non-breeding individuals. Consequently, the activity pattern does not seem to account for previously reported differences in longevity between breeding and non-breeding animals.     

Tracking of human cells in mice

Tracking and tracing of transplanted cells in mice is required in many fields of research. Examples are transplantation of stem cells into organs of mice to study their differentiation capacity and injection of tumor cells to examine metastatic behavior. In the present study we tested the lipid dye CM-DiI and red fluorescent nanoparticles Qdot655 for their applicability in tagging and tracing of human cells in mice. Labeling of different cell types, including MCF-7 human breast cancer cells, human cord blood derived cells, human NeoHep cells and human hepatopancreatic precursor cells, is technically easy and did not compromise further cell culture. After transplantation of CM-DiI or Qdot655 marked cells, red fluorescent structures could be detected already in unprocessed paraffin slices of the studied organs, namely liver, lung, pancreas, kidney, spleen and bone marrow. Next, we examined whether the red fluorescent structures represent the transplanted human cells. For this purpose, we established an in situ hybridization (ISH) technique that allows clear-cut differentiation between human and murine nuclei, based on simultaneous hybridization with human alu and mouse major satellite (mms) probes. We observed a high degree of coincidence between CM-DiI-marked cells and alu positive nuclei. However, also some mms positive cells contained CM-DiI, suggesting phagocytosis of the transplanted CM-DiI-marked cells. The degree of such CM-DiI-positive mouse cells depended on the cell type and route of administration. From a technical point of view it was important that CM-DiI-positive structures in paraffin slices remained fluorescent also after ISH. In contrast, Qdot655 positive structures faded during further staining procedures. In conclusion, marking of cells with CM-DiI or Qdot655 prior to transplantation facilitates recovery of human cells, since a high fraction of positive structures in the host's tissue originate from the transplanted cells. However, CM-DiI or Qdot655 positive staining of individual cells in transplanted tissues is not sufficient to prove their human origin. Additional procedures, such as ISH with alu-probes, are essential, when characterizing individual cells.     

Differences in hippocampal protein expression at 3 days, 3 weeks, and 3 months following induction of perinatal asphyxia in the rat

Perinatal asphyxia (PA) was induced in Sprague-Dawley rats; pups were sacrificed 3 days, 3 weeks, and 3 months following the asphyctic insult, and hippocampal protein levels were determined by a gel-based proteomic method. Levels of antioxidant, metabolic, cytoskeleton, signaling, channel, proteasomal, chaperone, splicing, and synaptosomal proteins were dysregulated depending on the age following induction of PA. These proteins are proposed to be potential markers or pharmacological targets for PA.     

Targeting of mitochondria by 10-N-alkyl acridine orange analogues: role of alkyl chain length in determining cellular uptake and localization

10-N-Nonyl acridine orange (NAO) is used as a mitochondrial probe because of its high affinity for cardiolipin (CL). Targeting of NAO may also depend on mitochondrial membrane potential. As the nonyl group has been considered essential for targeting, a systematic study of alkyl chain length was undertaken; three analogues (10-methyl-, 10-hexyl-, and 10-hexadecyl-acridine orange) were synthesized and their properties studied in phospholipid monolayers and breast cancer cells. The shortest and longest alkyl chains reduced targeting, whereas the hexyl group was superior to the nonyl group, allowing very clear and specific targeting to mitochondria at concentrations of 20-100 nM, where no evidence of toxicity was apparent. Additional studies in wild-type and cardiolipin-deficient yeast cells suggested that cellular binding was not absolutely dependent upon cardiolipin.     

Quality control in biosynthetic pathways of N-linked glycoproteins in the yeast endoplasmic reticulum

Summary The initial steps in N-glycosylation involve the synthesis of dolichol-linked Glc₃Man₉GlcNAc₂ oligosaccharides and the transfer of these oligosaccharides to nascent polypeptides. These processes take place at the membrane of the endoplasmic reticulum (ER) and are conserved among eukaryotes. Once transferred to the protein the N-linked oligosaccharides are immediately trimmed by glycosidases located in the ER. This review focuses on the N-linked glycosylation pathway in the ER ofSaccharomyces cerevisiae andSchizosaccharomyces pombe. In particular, we outline how yeast cells ensure that only completely assembled lipid-linked oligosaccharides are transferred to nascent polypeptides. We will discuss the oligosaccharide trimming of glycoproteins with respect to glycoprotein quality control and degradation, focusing on the two different quality control mechanisms ofS. cerevisiae andS. pombe.Abbreviations CPY carboxypeptidase Y - ER endoplasmic reticulum - LLO lipid-linked oligosaccharide - NLO protein-linked oligosaccharide - OTase oligosaccharyltransferase     

An Integrated Model of Multiple-Condition ChIP-Seq Data Reveals Predeterminants of Cdx2 Binding

Regulatory proteins can bind to different sets of genomic targets in various cell types or conditions. To reliably characterize such condition-specific regulatory binding we introduce MultiGPS, an integrated machine learning approach for the analysis of multiple related ChIP-seq experiments. MultiGPS is based on a generalized Expectation Maximization framework that shares information across multiple experiments for binding event discovery. We demonstrate that our framework enables the simultaneous modeling of sparse condition-specific binding changes, sequence dependence, and replicate-specific noise sources. MultiGPS encourages consistency in reported binding event locations across multiple-condition ChIP-seq datasets and provides accurate estimation of ChIP enrichment levels at each event. MultiGPS''s multi-experiment modeling approach thus provides a reliable platform for detecting differential binding enrichment across experimental conditions. We demonstrate the advantages of MultiGPS with an analysis of Cdx2 binding in three distinct developmental contexts. By accurately characterizing condition-specific Cdx2 binding, MultiGPS enables novel insight into the mechanistic basis of Cdx2 site selectivity. Specifically, the condition-specific Cdx2 sites characterized by MultiGPS are highly associated with pre-existing genomic context, suggesting that such sites are pre-determined by cell-specific regulatory architecture. However, MultiGPS-defined condition-independent sites are not predicted by pre-existing regulatory signals, suggesting that Cdx2 can bind to a subset of locations regardless of genomic environment. A summary of this paper appears in the proceedings of the RECOMB 2014 conference, April 2–5.     

Persistence of the Attractiveness of Two Sex-specific Scents in Meadow Voles,Microtus pennsylvanicus

The function of an odour may be reflected in its fade-out time in the environment. In this study, we investigated fade-out times of two specific odours, the anogenital area scent and that of the posterolateral region. These two odours support opposite-sex preferences in male and female meadow voles, Microtus pennsylvanicus, but convey nonidentical information to conspecifics during the breeding season. The first experiment tested whether meadow voles respond preferentially to scents that were aged for 15 min (fresh) to 30 d. Males preferred female anogenital area scent to male anogenital area scent if both scents were ≤ 10 d old. By comparison, females preferred male anogenital area scent to female anogenital area scent if the scents were ≤ 25 d old. However, male and female voles preferred the posterolateral scent of males to that of females if the scents were ≤ 1 d old. Thus, fade-out times for these two scents differ for males and females, suggesting different functions. In the second experiment, male and female voles preferred fresh anogenital area scent and fresh posterolateral region scent compared with those same scents that were older. This result suggests that older scents may have lost information over time about the sex of the donor. Overall, data from both experiments indicate that voles may use specific scents for communication in different social contexts.     

GPI anchor biosynthesis in yeast: phosphoethanolamine is attached to the alpha1,4-linked mannose of the complete precursor glycophospholipid

Cells synthesize the GPI anchor carbohydrate core by successively adding N-acetylglucosamine, three mannoses, and phosphoethanolamine (EtN-P) onto phosphatidylinositol, thus forming the complete GPI precursor lipid which is then added to proteins. Previously, we isolated a GPI deficient yeast mutant accumulating a GPI intermediate containing only two mannoses, suggesting that it has difficulty in adding the third, alpha1,2-linked Man of GPI anchors. The mutant thus displays a similar phenotype as the mammalian mutant cell line S1A-b having a mutation in the PIG-B gene. The yeast mutant, herein named gpi10-1 , contains a mutation in YGL142C, a yeast homolog of the human PIG-B. YGL142C predicts a highly hydrophobic integral membrane protein which by sequence is related to ALG9, a yeast gene required for adding Man in alpha1,2 linkage to N-glycans. Whereas gpi10-1 cells grow at a normal rate and make normal amounts of GPI proteins, the microsomes of gpi10-1 are completely unable to add the third Man in an in vitro assay. Further analysis of the GPI intermediate accumulating in gpi10 shows it to have the structure Manalpha1-6(EtN-P-)Manalpha1-4GlcNalpha1- 6(acyl) Inositol-P-lipid. The presence of EtN-P on the alpha1,4-linked Man of GPI anchors is typical of mammalian and a few other organisms but had not been observed in yeast GPI proteins. This additional EtN-P is not only found in the abnormal GPI intermediate of gpi10-1 but is equally present on the complete GPI precursor lipid of wild type cells. Thus, GPI biosynthesis in yeast and mammals proceeds similarly and differs from the pathway described for Trypanosoma brucei in several aspects.