Multi-isotopic analysis reveals individual mobility and diet at the Early Iron Age monumental tumulus of Magdalenenberg, Germany

For the Early Iron Age western Hallstatt culture, which includes the site of Magdalenenberg in southwest Germany, it has been proposed that people were mobile and maintained far reaching social and trading networks throughout Europe. We tested this hypothesis by analyzing multiple isotopes (strontium, oxygen, sulfur, carbon, and nitrogen) of the preserved skeletons from the Magdalenenberg elite cemetery to determine diets and to look for evidence of mobility. The analysis of carbon, nitrogen, and sulfur isotope ratios in collagen of humans (n = 50) and associated domestic fauna (n = 10) indicates a terrestrial-based diet. There was a heterogeneous range of isotope values in both strontium (0.70725 to 0.71923, n = 76) and oxygen (13.4‰ to 18.5‰, n = 78) measured in tooth enamel. Although many of the individuals had values consistent with being from Hallstatt culture sites within southwest Germany, some individuals likely originated from further afield. Possible areas include the Alps of Switzerland and Austria or even locations in Italy. Our study strongly supports the assumption of far reaching social and economic networks in the western Hallstatt culture.     

Permanent tooth mineralization in bonobos (Pan paniscus) and chimpanzees (P. troglodytes)

The timing of tooth mineralization in bonobos (Pan paniscus) is virtually uncharacterized. Analysis of these developmental features in bonobos and the possible differences with its sister species, the chimpanzee (P. troglodytes), is important to properly quantify the normal ranges of dental growth variation in closely related primate species. Understanding this variation among bonobo, chimpanzee and modern human dental development is necessary to better contextualize the life histories of extinct hominins. This study tests whether bonobos and chimpanzees are distinguished from each other by covariance among the relative timing and sequences of tooth crown initiation, mineralization, root extension, and completion. Using multivariate statistical analyses, we compared the relative timing of permanent tooth crypt formation, crown mineralization, and root extension between 34 P. paniscus and 80 P. troglodytes mandibles radiographed in lateral and occlusal views. Covariance among our 12 assigned dental scores failed to statistically distinguish between bonobos and chimpanzees. Rather than clustering by species, individuals clustered by age group (infant, younger or older juvenile, and adult). Dental scores covaried similarly between the incisors, as well as between both premolars. Conversely, covariance among dental scores distinguished the canine and each of the three molars not only from each other, but also from the rest of the anterior teeth. Our study showed no significant differences in the relative timing of permanent tooth crown and root formation between bonobos and chimpanzees. Am J Phys Anthropol, 2012. © 2012 Wiley Periodicals, Inc.     

Role of EscP (Orf16) in Injectisome Biogenesis and Regulation of Type III Protein Secretion in Enteropathogenic Escherichia coli

Enteropathogenic Escherichia coli employs a type III secretion system (T3SS) to translocate virulence effector proteins directly into enterocyte host cells, leading to diarrheal disease. The T3SS is encoded within the chromosomal locus of enterocyte effacement (LEE). The function of some of the LEE-encoded proteins remains unknown. Here we investigated the role of the Orf16 protein in T3SS biogenesis and function. An orf16 deletion mutant showed translocator and effector protein secretion profiles different from those of wild-type cells. The orf16 null strain produced T3S structures with abnormally long needles and filaments that caused weak hemolysis of red blood cells. Furthermore, the number of fully assembled T3SSs was also reduced in the orf16 mutant, indicating that Orf16, though not essential, is required for efficient T3SS assembly. Analysis of protein secretion revealed that Orf16 is a T3SS-secreted substrate and regulates the secretion of the inner rod component EscI. Both pulldown and yeast two-hybrid assays showed that Orf16 interacts with the C-terminal domain of an inner membrane component of the secretion apparatus, EscU; the inner rod protein EscI; the needle protein EscF; and the multieffector chaperone CesT. These results suggest that Orf16 regulates needle length and, along with EscU, participates in a substrate specificity switch from early substrates to translocators. Taken together, our results suggest that Orf16 acts as a molecular measuring device in a way similar to that of members of the Yersinia YscP and flagellar FliK protein family. Therefore, we propose that this protein be renamed EscP.     

Okazaki fragment processing-independent role for human Dna2 enzyme during DNA replication

Dna2 is an essential helicase/nuclease that is postulated to cleave long DNA flaps that escape FEN1 activity during Okazaki fragment (OF) maturation in yeast. We previously demonstrated that the human Dna2 orthologue (hDna2) localizes to the nucleus and contributes to genomic stability. Here we investigated the role hDna2 plays in DNA replication. We show that Dna2 associates with the replisome protein And-1 in a cell cycle-dependent manner. Depletion of hDna2 resulted in S/G(2) phase-specific DNA damage as evidenced by increased γ-H2AX, replication protein A foci, and Chk1 kinase phosphorylation, a readout for activation of the ATR-mediated S phase checkpoint. In addition, we observed reduced origin firing in hDna2-depleted cells consistent with Chk1 activation. We next examined the impact of hDna2 on OF maturation and replication fork progression in human cells. As expected, FEN1 depletion led to a significant reduction in OF maturation. Strikingly, the reduction in OF maturation had no impact on replication fork progression, indicating that fork movement is not tightly coupled to lagging strand maturation. Analysis of hDna2-depleted cells failed to reveal a defect in OF maturation or replication fork progression. Prior work in yeast demonstrated that ectopic expression of FEN1 rescues Dna2 defects. In contrast, we found that FEN1 expression in hDna2-depleted cells failed to rescue genomic instability. These findings suggest that the genomic instability observed in hDna2-depleted cells does not arise from defective OF maturation and that hDna2 plays a role in DNA replication that is distinct from FEN1 and OF maturation.     

Exploring the Unique N-Glycome of the Opportunistic Human Pathogen Acanthamoeba

Glycans play key roles in host-pathogen interactions; thus, knowing the N-glycomic repertoire of a pathogen can be helpful in deciphering its methods of establishing and sustaining a disease. Therefore, we sought to elucidate the glycomic potential of the facultative amoebal parasite Acanthamoeba. This is the first study of its asparagine-linked glycans, for which we applied biochemical tools and various approaches of mass spectrometry. An initial glycomic screen of eight strains from five genotypes of this human pathogen suggested, in addition to the common eukaryotic oligomannose structures, the presence of pentose and deoxyhexose residues on their N-glycans. A more detailed analysis was performed on the N-glycans of a genotype T11 strain (4RE); fractionation by HPLC and tandem mass spectrometric analyses indicated the presence of a novel mannosylfucosyl modification of the reducing terminal core as well as phosphorylation of mannose residues, methylation of hexose and various forms of pentosylation. The largest N-glycan in the 4RE strain contained two N-acetylhexosamine, thirteen hexose, one fucose, one methyl, and two pentose residues; however, in this and most other strains analyzed, glycans with compositions of Hex_8–9HexNAc₂Pnt_0–1 tended to dominate in terms of abundance. Although no correlation between pathogenicity and N-glycan structure can be proposed, highly unusual structures in this facultative parasite can be found which are potential virulence factors or therapeutic targets.     

Protein kinase C (PKC)ζ-mediated Gαq stimulation of ERK5 protein pathway in cardiomyocytes and cardiac fibroblasts

Gq-coupled G protein-coupled receptors (GPCRs) mediate the actions of a variety of messengers that are key regulators of cardiovascular function. Enhanced Gα(q)-mediated signaling plays an important role in cardiac hypertrophy and in the transition to heart failure. We have recently described that Gα(q) acts as an adaptor protein that facilitates PKCζ-mediated activation of ERK5 in epithelial cells. Because the ERK5 cascade is known to be involved in cardiac hypertrophy, we have investigated the potential relevance of this pathway in cardiovascular Gq-dependent signaling using both cultured cardiac cell types and chronic administration of angiotensin II in mice. We find that PKCζ is required for the activation of the ERK5 pathway by Gq-coupled GPCR in neonatal and adult murine cardiomyocyte cultures and in cardiac fibroblasts. Stimulation of ERK5 by angiotensin II is blocked upon pharmacological inhibition or siRNA-mediated silencing of PKCζ in primary cultures of cardiac cells and in neonatal cardiomyocytes isolated from PKCζ-deficient mice. Moreover, upon chronic challenge with angiotensin II, these mice fail to promote the changes in the ERK5 pathway, in gene expression patterns, and in hypertrophic markers observed in wild-type animals. Taken together, our results show that PKCζ is essential for Gq-dependent ERK5 activation in cardiomyocytes and cardiac fibroblasts and indicate a key cardiac physiological role for the Gα(q)/PKCζ/ERK5 signaling axis.     

Simultaneous acquisition of PAR and PAIN spectra

We present a scheme that allows the simultaneous detection of PAR and PAIN correlation spectra in a single two-dimensional experiment. For both spectra, we obtain almost the same signal-to-noise ratio as if a PAR or PAIN spectrum is recorded separately, which in turn implies that one of the spectra may be considered additional information for free. The experiment is based on the observation that in a PAIN experiment, the PAR condition is always also fulfilled. The performance is demonstrated experimentally using uniformly ¹³C,¹⁵N-labeled samples of N–f–MLF–OH and ubiquitin.     

Cytopathology of Tomato torrado virus Infection in Tomato and Nicotiana benthamiana

Electron microscopy studies were carried out to investigate the cytopathological changes induced in tomato leaves by Tomato torrado virus (ToTV) that infects tomato plants worldwide causing severe necrotic symptoms. Plants infected with one of the Polish isolates of ToTV were used for cytopathological research. The results revealed severe cellular alterations, especially in Solanum lycopersicum. Moreover, it was shown that crystalline aggregates of virions occurred not only within the phloem cells as it has been previously reported.     

Temperature scans/cycles for the detection of low abundant DNA point mutations on microarrays

The possibility to detect low abundant DNA point mutations is essential for early cancer diagnosis and/or prognosis. Furthermore, in order to be less invasive, the somatic mutations are not only sought in tumor extract samples but also from body fluids or stools rendering their content even more diluted compared to the wild type sequences. In this short communication, we propose two protocols based on temperature scans or cycles for the enrichment of the mutation strands hybridized on microarrays. We predict numerically and confirm experimentally a 10-fold increase in the fraction of mutated DNA hybridized on the microarray compared to the sample content. Coupled to more standard solution phase enrichment techniques, it would be possible to lower by one order of magnitude the current detection limit with the advantage of multiple mutation detections offered by the microarray technology.